Intermediate for the manufacture of glucagon and GLP-1 dual agonists and method for the manufacture thereof

Abstract

A method useful for the preparation of novel intermediates and mazdutide or pharmaceutically acceptable salts thereof is provided. In this method, instead of stepwise coupling of Fmoc-Gly-OH, coupling of Fmoc-Gly-Gly-OH is used, and by directly coupling the OSU active ester of the side chain synthesized by DIC / HOSu to the peptide chain, the reaction time is shortened and possible racemization and impurity generation are reduced, thereby reducing the number of steps while maintaining and improving quality and purity.

Description

Technology Field

[0001] Cross-reference of related applications

[0002] This application claims priority to Chinese application No. 202311494392.7 filed on November 10, 2023, the entirety of which is incorporated herein by reference.

[0003] Technology field

[0004] The present invention relates to an intermediate or a pharmaceutically acceptable salt thereof for producing a compound that acts on both glucagon (Gcg) receptors and glucagon-like peptide-1 (GLP-1) receptors, and specifically to an intermediate or a pharmaceutically acceptable salt thereof for producing mazdutide, an oxintomodulin / glucagon analog, and a method for producing the same. Background Technology

[0005] Mazdutide is a Gcg and GLP-1 dual agonist peptide that can be used to treat T2D and obesity, and is an oxintomodulin / glucagon analog consisting of a main peptide chain and a side chain containing 34 amino acids, wherein 2-aminoisobutyric acid (Aib) at position 2 is a non-natural amino acid and the C-terminus is an amide. The side chain consists of two 2-(2-(2-aminoethoxy)ethoxy)acetic acid [2-(2-(2-aminoethoxy)ethoxy)acetic acid, AEEA] residues, one γ-Glu residue, and one eicosaneate residue, and this side chain is bound to the Lys20 site of the main peptide chain through the two 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) residues. A method for preparing such Gcg and GLP-1 dual agonist peptides is described in the prior art.

[0006] However, there is still a need for improved methods to produce more cost-effective Gcg and GLP-1 dual-agonist peptides that enable the generation of peptides with various advantages, including commercially required purity. At the same time, there is also a need for more rapid and stable methods involving stable compounds to provide Gcg and GLP-1 dual-agonist peptides through fewer or simpler manufacturing and / or purification steps. It is well known that there are several technical challenges in the production of these Gcg and GLP-1 dual-agonist peptides that can affect overall yield and purity.

[0007] In the present invention, instead of coupling Fmoc-Gly-OH stepwise in the solid-phase synthesis of peptides, the coupling of Fmoc-Gly-Gly-OH is used, and by directly coupling the OSU active ester of the side chain synthesized by DIC / HOSu to the peptide chain, the reaction time is shortened and possible racemization and impurity generation are reduced, thereby reducing the number of steps while maintaining and improving quality and purity.

[0008] In one embodiment, the present invention provides a method for preparing a compound of the following chemical formula (sequence number 1).

[0009]

[0010] The method includes solid-phase synthesis of compounds, but,

[0011] Fmoc-Gly-Gly-OH is used in the corresponding solid-phase synthesis, and

[0012] R1 and R3 are base-stabilizing side chain protectors, and

[0013] R2 is an ivDde side chain protector.

[0014] In one embodiment, the present invention provides a method for preparing a compound of the following chemical formula (sequence number 2).

[0015] H-Aib-QGTFTSDYSKYLDEKKAKEFV-EWLLEGGSSG-NH2

[0016] The ε-amino group of the Lys side chain at position 20 is ([2-(2-aminoethoxy)-ethoxy]-acetyl)-2-(γ-Glu)-CO-(CH2) 18 It is chemically modified by conjugating to CO2H, and

[0017] The method is

[0018] (i) A solid-phase synthesis step of a compound of the following chemical formula (Sequence No. 1), wherein

[0019]

[0020] Fmoc-Gly-Gly-OH is used in the corresponding solid-phase synthesis, and

[0021] R1 and R3 are base-stabilizing side chain protectors, and

[0022] R2 is the ivDde side chain protector, phase,

[0023] (ii) tBuO-C obtained through a selective deprotection step of Lys at position 20 and DIC / HOSu 20 A step of directly coupling the -γGlu(tBu)-AEEA-AEEA-OSu active ester to the ε-amino group of the Lys side chain at position 20,

[0024] (iii) a step of cleaving the corresponding compound on a solid support and removing the base stability side chain protector, and

[0025] (iv) A method including a purification step of the corresponding compound.

[0026] In another aspect, the present application provides a compound of the following formula I (SEQ ID NO. 3) or a pharmaceutically acceptable salt thereof, a compound of the following formula II (SEQ ID NO. 4) or a pharmaceutically acceptable salt thereof, or a compound of the following formula III (SEQ ID NO. 5) or a pharmaceutically acceptable salt thereof:

[0027]

[0028] Chemical formula I,

[0029]

[0030] Chemical formula II,

[0031]

[0032] Chemical formula III.

[0033] In some embodiments, the compound of Formula I or its pharmaceutically acceptable salt, the compound of Formula II or its pharmaceutically acceptable salt, or the compound of Formula III or its pharmaceutically acceptable salt is a compound or its pharmaceutically acceptable salt selected from the following compounds or their pharmaceutically acceptable salts:

[0034]

[0035] Compound 2 (Sequence No. 6),

[0036]

[0037] Compound 3 (Sequence No. 7),

[0038]

[0039] Compound 4 (Sequence No. 8).

[0040] In another aspect, the present application provides a compound of the following chemical formula (Sequence No. 1), and

[0041]

[0042] The said compound is prepared by a method including solid-phase synthesis of the said compound,

[0043] Fmoc-Gly-Gly-OH is used in the corresponding solid-phase synthesis, and

[0044] R1 and R3 are base-stabilizing side chain protectors, and

[0045] R2 is an ivDde side chain protector.

[0046] In another aspect, the present application provides a compound of the following chemical formula (Sequence No. 2), and

[0047] H-Aib-QGTFTSDYSKYLDEKKAKEFV-EWLLEGGSSG-NH2

[0048] The ε-amino group of the Lys side chain at position 20 is ([2-(2-aminoethoxy)-ethoxy]-acetyl)-2-(γ-Glu)-CO-(CH2) 18 It is chemically modified by conjugating to CO2H, and

[0049] The compound in question is

[0050] (i) A solid-phase synthesis step of a compound of the following chemical formula (Sequence No. 1), wherein

[0051]

[0052] Fmoc-Gly-Gly-OH is used in the corresponding solid-phase synthesis, and

[0053] R1 and R3 are base-stabilizing side chain protectors, and

[0054] R2 is the ivDde side chain protector, phase,

[0055] (ii) tBuO-C obtained through a selective deprotection step of Lys at position 20 and DIC / HOSu 20 A step of directly coupling the -γGlu(tBu)-AEEA-AEEA-OSu active ester to the ε-amino group of the Lys side chain at position 20,

[0056] (iii) a step of cleaving the corresponding compound on a solid support and removing the base stability side chain protector, and

[0057] (iv) It is prepared by a method including a purification step of the corresponding compound. Specific details for implementing the invention

[0058] Specific embodiments of the present invention are described in detail below. The specific embodiments described herein should be understood as not limiting the invention, but merely as illustrative and illustrative. The ranges and boundary values ​​of any values ​​disclosed herein are not limited to exact ranges or values, and should be understood as encompassing values ​​close to these ranges or values. In the case of numerical ranges, the boundary values ​​of each range, the boundary values ​​of each range and single point value, as well as the single point values, may be combined with each other to obtain one or more new numerical ranges, which should be interpreted as specifically disclosed herein.

[0059] The present invention provides a method for preparing a compound of the following chemical formula (Sequence No. 1), and

[0060]

[0061] The method includes solid-phase synthesis of the said compound, but,

[0062] Fmoc-Gly-Gly-OH is used in the corresponding solid-phase synthesis, and

[0063] R1 and R3 are base-stabilizing side chain protectors, and

[0064] R2 is an ivDde side chain protector.

[0065] The method involves chemical formula A, Fmoc-Gly-Gly-OH bonded to the solid phase, It includes a step of coupling to the peptide sequence of. The coupling step comprises chemical formula B bound to the solid phase, Generates the peptide sequence of.

[0066] In some embodiments, solid-phase synthesis is performed on an Fmoc amide resin solid support.

[0067] In a preferred embodiment, the Fmoc amide resin is a cost-reducing Rink amide resin.

[0068] Various base stability protecting groups are known in the art and may be used in the method of the present invention. In one embodiment of the present invention, the R1 protecting group used in the synthesis of the compound is (a) tert-butoxycarbonyl (Boc) for Trp and Lys, (b) tert-butyl ester (OtBu) for Asp and Glu, (c) tert-butyl (tBu) for Ser, Thr and Tyr, (d) triphenylmethyl [trityl, Trt] for Gln, and (e) Trt for His. In some embodiments, the R3 protecting group for His is bonded to the amino group of His, and R3 is Boc. In a preferred embodiment of the method of the present invention, R1 is Boc for Trp and Lys, OtBu for Asp and Glu, tBu for Ser, Thr and Tyr, and Trt for Gln and His.

[0069] The solid-phase synthesis of the compound is performed on a Fmoc amide resin solid support, and the first step is a sequential coupling step of Fmoc amino acids of the peptide following the Fmoc deprotection step of the amide resin. In the present invention, the risk of Gly impurity formation is reduced by performing Gly-Gly coupling at positions 29–30 of the peptide chain using the coupling of Fmoc-Gly-Gly-OH instead of stepwise coupling of Fmoc-Gly-OH. In addition, material costs are reduced by replacing the coupling of the glycine-threonine pseudoproline dipeptide with the stepwise coupling of Gly and Thr at positions 4 and 5, and no peptides with corresponding amino acids deleted are found in the crude peptide obtained by the process of the present invention.

[0070] Specifically, solid-phase synthesis is performed on a Fmoc amide resin solid support, and the Fmoc deprotection step of the amide resin and the following Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH·H2O, Fmoc-Gly-Gly-OH, Fmoc-Glu(OtBu)-OH·H2O, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(OtBu)-OH·H2O, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH·H2O, Fmoc-Lys(ivDde)-OH, Fmoc-Ala-OH·H2O, Fmoc-Lys(Boc)-OH, It includes sequential coupling steps of Fmoc-Lys(Boc)-OH, Fmoc-Glu(OtBu)-OH·H2O, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Gln(Trt)-OH, Fmoc-Aib-OH, and Boc-His(Trt)-OH.

[0071] In some embodiments, solid-phase synthesis is performed on a Fmoc amide resin solid support and includes a Fmoc deprotection step of the amide resin prior to the coupling step of Fmoc-Gly-Gly-OH and a sequential coupling step of Fmoc amino acids of the following Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, and Fmoc-Pro-OH·H2O. After the coupling of Fmoc-Gly-Gly-OH, the solid-phase synthesis is as follows: Fmoc-Glu(OtBu)-OH·H2O, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(OtBu)-OH·H2O, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH·H2O, Fmoc-Lys(ivDde)-OH, Fmoc-Ala-OH·H2O, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Glu(OtBu)-OH·H2O, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, It further includes sequential coupling steps of Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Gln(Trt)-OH, Fmoc-Aib-OH, and Boc-His(Trt)-OH.

[0072] In some embodiments, solid-phase synthesis is performed on a Fmoc amide resin solid support, and the Fmoc deprotection step of the amide resin and the following Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH·H2O, Fmoc-Gly-Gly-OH, Fmoc-L-Glu(OtBu)-OH·H2O, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-L-Glu(OtBu)-OH·H2O, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-L-Glu(OtBu)-OH·H2O, Fmoc-Lys(ivDde)-OH, Fmoc-Ala-OH·H2O, Fmoc-Lys(Boc)-OH, It includes sequential coupling steps of Fmoc-Lys(Boc)-OH, Fmoc-L-Glu(OtBu)-OH·H2O, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Gln(Trt)-OH, Fmoc-Aib-OH, and Boc-His(Trt)-OH.

[0073] In some embodiments, solid-phase synthesis is performed on a Fmoc amide resin solid support and includes a sequential coupling step of Fmoc amino acids of the following Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, and Fmoc-Pro-OH·H2O prior to the Fmoc deprotection step of the amide resin and the coupling step of Fmoc-Gly-Gly-OH. After the coupling step of Fmoc-Gly-Gly-OH, the solid-phase synthesis is as follows: Fmoc-L-Glu(OtBu)-OH·H2O, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-L-Glu(OtBu)-OH·H2O, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-L-Glu(OtBu)-OH·H2O, Fmoc-Lys(ivDde)-OH, Fmoc-Ala-OH·H2O, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-L-Glu(OtBu)-OH·H2O, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, It further includes sequential coupling steps of Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Gln(Trt)-OH, Fmoc-Aib-OH, and Boc-His(Trt)-OH.

[0074] In a preferred embodiment, naturally occurring amino acids are preferably used in this application.

[0075] SPPS comprises several basic steps that are repeated as the peptide chain is extended by adding additional amino acids to the elongating peptide chain. The 'solid phase' refers to resin particles [e.g., link-amide resins] to which the elongating peptide chain is attached following the original amino acid. Since the chain is attached to the particles, they can be treated as a set of solid particles (particularly in washing and separation steps, e.g., filtration steps).

[0076] The repeated steps of SPPS include deprotection, activation, and coupling steps.

[0077] (1) Deprotection step: The final acid on the peptide chain is maintained in a 'protected' state before the start of each cycle. As used herein, the term 'protected' means that a protecting group is attached at a specific location, that is, the 'amino' terminus of the peptide chain is attached to a functional group that protects the acid from unwanted reactions. Various protecting groups are well known, and alternative protecting groups may be suitable for a specific method. When the next amino acid is added, the 'protecting group' is removed ('deprotection' step).

[0078] (2) Activation step: A compound ('activator') is added to the reaction to produce intermediate amino acid species that are more likely to couple with acid after deprotection on the peptide chain.

[0079] (3) Coupling step: The species after activation is coupled to the existing peptide chain.

[0080] The reagent for deprotection may be one commonly used in the field. In the present invention, it is preferable to use a Pip / DMF / NMP solution or a DBU deprotection solution, wherein Pip may be 15% to 20% by weight, and the mass ratio of DMF and NMP is preferably 1:(0.9 to 1.1). For example, preferably, the mass ratio of DMF, Pip, and NMP is 2.2:1:2.4.

[0081] In a preferred embodiment, the deprotection reaction in the solid-phase synthesis of the present invention comprises the step of performing deprotection twice for 13 to 17 minutes (e.g., 14 to 16 minutes or 15 minutes) each in the first 10 solid-phase synthesis cycles, and the step of performing deprotection twice for 25 to 35 minutes (e.g., 29 to 31 minutes or 30 minutes) each in the last 13 to 33 solid-phase synthesis cycles. Reducing the time and frequency of deprotection can shorten the production period and reduce material costs.

[0082] In some embodiments, deprotection may be performed three times (for 15 minutes each) for nucleotide Val, and deprotection may also be performed three times (for 30 minutes each) for amino acid Phe at position 22 after Val. In some embodiments, deprotection may be performed four times (for 30 minutes each) for amino acids Thr at positions 5 and 7.

[0083] One of the most commonly used and studied activation methods for peptide synthesis is in carbodiimide-based applications. Carbodiimides contain two weakly basic nitrogen atoms, which react with the carboxylic acid of an amino acid derivative to form a highly reactive O-acylisourea compound. In a preferred embodiment, 1-hydroxybenzotriazole (HOBt) or 1-hydroxy-7-azabenzotriazole (HOAt) is introduced as an additive to the carbodiimide activation method to significantly improve the carbodiimide activation method. Instead of HOBt or HOAt, other additives, for example, 2-cyano-2-(hydroxyimino)ethyl acetate (Oxima) or 1-hydroxy-2,5-pyrrolidinedione (NHS) may be used.

[0084] In addition, onium salt condensation reagents can be used for activation, which are highly reactive and rapid. For example, 1 H -Benzotriazole-1-yloxytripyrrolidineyl hexafluorophosphate(1 H -benzotriazole-1-yloxytripyrrolidinyl hexafluorophosphate (PyBOP), etc., may be used. For the use of reagents, diisopropylethylamine (DIEA) and to activate amino acids are used. N -methylmorpholine ( N The addition of organic bases such as methylmorpholine (NMM) is required. 1-hydroxybenzotriazole (HOBt) can also be introduced as an additive in the onium salt condensation reaction.

[0085] Activation can also be performed using an organophosphorus phosphate condensate, for example, 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4-one [3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4-one, DEPBT]. DEPBT is an organophosphorus phosphate condensate derived from HOBt and has advantages such as good solubility, mild reaction conditions, no need to protect partial amino acid side chains, and a low racemization rate. An organic base, for example, DIEA, can be added to the reaction of the reagent.

[0086] In the method of the present invention, a preferred activation reaction system is DIC / HOBt / HOAt / Oxyma in DMF or DMF / NMP, or PyBOP / HOBt in DMF or DMF / NMP, or DEPBT in DMF or DMF / NMP.

[0087] In one embodiment of the present invention, the molar amount of the activator may be 3 to 7 times greater than the molar amount of the resin. Additionally, the ratio of the molar amount of the additive to the molar amount of the activator may be about 1. If the addition of an organic base is required, the amount of the organic base used is preferably the sum of the molar amounts of the activator and any additive.

[0088] In a preferred embodiment, the coupling step of the first 11 amino acids (i.e., the first 10 solid-phase synthesis cycles) is performed using a low equivalent amount (e.g., 2 to 4 equivalents, preferably 2, 3, or 4 equivalents) of Fmoc amino acids relative to the resin, and the coupling step of the remaining amino acids is performed using a high equivalent amount (e.g., 4 to 6 equivalents, e.g., 4, 5, or 6 equivalents) of Fmoc amino acids relative to the resin. In some embodiments, the coupling step at His at position 1 and Lys at position 20 may be performed using 3 equivalents of Fmoc amino acids. In some embodiments, the coupling step may be performed at a temperature of 20 to 30 °C for about 3 hours, e.g., 2 hours 50 minutes to 3 hours 10 minutes. The reaction time may be adjusted according to the degree of reaction completion, but the total reaction time must be ≤12 hours.

[0089] More generally, in relation to the method of the present invention, the SPPS structure is preferably achieved by a standard Fmoc peptide chemistry technique using sequential coupling with an automated peptide synthesizer. The preferred resin is a link amide resin. DMF / NMP is a preferred solvent system, and DMF is used for swelling of the resin. A Pip / DMF / NMP solution is preferably used for deprotection of the resin. A Pip / DMF / NMP solution or a DBU deprotection solution is preferably used for subsequent Fmoc deprotection treatment, which is treated for 2 x 15 minutes during the first 10 solid-phase synthesis cycles, preferably for 2 x 30 minutes during the subsequent coupling step of amino acids, and may also be treated for 2 x 30 minutes in the case of a difficult coupling step. After deprotection, the resin is preferably washed for 8 x 1 to 3 minutes with 7 to 9 (preferably 8) times the amount of DMF. The amino acid activation step is preferably performed at room temperature (e.g., 20–30 °C) or 0–10 °C for 30 minutes using an activator / DMF / NMP solution optionally containing an additive or an inorganic base. The activation step is performed for 1–6 minutes under stirring. For each individual amino acid, a coupling step of the activated amino acid and the peptide bound to the resin is performed for a specific time. After each coupling step, 7–9 (preferably 8) times the amount of DMF washing solvent is preferably performed for 8 × 1–3 minutes.

[0090] Other cleaning agents, such as DCM and NMP, may also be used for the addition of Val, and the washing step may be performed as many times as necessary.

[0091] The difficult coupling step can also be performed by refeeding, for example, by repeating the activation, coupling, and washing steps after washing, or by using a different activator during refeeding. Solid-phase synthesis optionally further includes a terminal capping step after the coupling reaction to block unreacted amino groups or acetylate the N-terminus of the intact peptide.

[0092] The present invention provides a method for preparing a compound of the following chemical formula (sequence number 2), and

[0093] H-Aib-QGTFTSDYSKYLDEKKAKEFV-EWLLEGGSSG-NH2

[0094] The ε-amino group of the Lys side chain at position 20 is ([2-(2-aminoethoxy)-ethoxy]-acetyl)-2-(γ-Glu)-CO-(CH2) 18 It is chemically modified by conjugating to CO2H, and

[0095] The method is

[0096] (i) A solid-phase synthesis step of a compound of the following chemical formula (Sequence No. 1), wherein

[0097]

[0098] Fmoc-Gly-Gly-OH is used in the corresponding solid-phase synthesis, and

[0099] R1 and R3 are base-stabilizing side chain protectors, and

[0100] R2 is the ivDde side chain protector, phase,

[0101] (ii) tBuO-C obtained through a selective deprotection step of Lys at position 20 and DIC / HOSu 20 A step of directly coupling the -γGlu(tBu)-AEEA-AEEA-OSu active ester to the ε-amino group of the Lys side chain at position 20,

[0102] (iii) a step of cleaving the corresponding compound on a solid support and removing the base stability side chain protector, and

[0103] (iv) A method including a purification step of the corresponding compound.

[0104] In some embodiments, solid-phase synthesis is performed on an Fmoc amide resin solid support.

[0105] In a preferred embodiment, Fmoc amide resin is a cost-reducing link amide resin.

[0106] Various base stability protecting groups are known in the art and may be used in the method of the present invention. In one embodiment of the present invention, the R1 protecting group of the base stability side chain used in the synthesis of the compound is (a) tert-butoxycarbonyl (Boc) for Trp and Lys, (b) tert-butyl ester (OtBu) for Asp and Glu, (c) tert-butyl (tBu) for Ser, Thr and Tyr, (d) triphenylmethyl (trityl, Trt) for Gln, and (e) Trt for His. In some embodiments, the R3 protecting group for His is bonded to the amino group of His, and R3 is Boc. In a preferred embodiment of the method of the present invention, R1 is Boc for Trp and Lys, OtBu for Asp and Glu, tBu for Ser, Thr and Tyr, and Trt for Gln and His.

[0107] The solid-phase synthesis of the compound is performed on a Fmoc amide resin solid support, and the first step is a sequential coupling step of Fmoc amino acids of the peptide following the Fmoc deprotection step of the amide resin. In the present invention, the risk of Gly impurity formation is reduced by performing Gly-Gly coupling at positions 29–30 of the peptide chain using the coupling of Fmoc-Gly-Gly-OH instead of stepwise coupling of Fmoc-Gly-OH. In addition, material costs are reduced by replacing the coupling of the glycine-threonine pseudoproline dipeptide with the stepwise coupling of Gly and Thr at positions 4 and 5, and no peptides with corresponding amino acids deleted are found in the crude peptide obtained by the process of the present invention.

[0108] Specifically, in step (i), the solid-phase synthesis of the compound is performed on a Fmoc amide resin solid support, and the Fmoc deprotection step of the amide resin and the following Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH·H2O, Fmoc-Gly-Gly-OH, Fmoc-Glu(OtBu)-OH·H2O, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(OtBu)-OH·H2O, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH·H2O, Fmoc-Lys(ivDde)-OH, Fmoc-Ala-OH·H2O, Fmoc-Lys(Boc)-OH, It includes sequential coupling steps of Fmoc-Lys(Boc)-OH, Fmoc-Glu(OtBu)-OH·H2O, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Gln(Trt)-OH, Fmoc-Aib-OH, and Boc-His(Trt)-OH.

[0109] In some embodiments, solid-phase synthesis is performed on a Fmoc amide resin solid support and includes a sequential coupling step of Fmoc amino acids of the following Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, and Fmoc-Pro-OH·H2O prior to the Fmoc deprotection step of the amide resin and the coupling step of Fmoc-Gly-Gly-OH. After the coupling step of Fmoc-Gly-Gly-OH, the solid-phase synthesis is as follows: Fmoc-L-Glu(OtBu)-OH·H2O, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-L-Glu(OtBu)-OH·H2O, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-L-Glu(OtBu)-OH·H2O, Fmoc-Lys(ivDde)-OH, Fmoc-Ala-OH·H2O, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-L-Glu(OtBu)-OH·H2O, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, It further includes sequential coupling steps of Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Gln(Trt)-OH, Fmoc-Aib-OH, and Boc-His(Trt)-OH.

[0110] In some embodiments, solid-phase synthesis is performed on a Fmoc amide resin solid support, and the Fmoc deprotection step of the amide resin and the following Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH·H2O, Fmoc-Gly-Gly-OH, Fmoc-L-Glu(OtBu)-OH·H2O, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-L-Glu(OtBu)-OH·H2O, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-L-Glu(OtBu)-OH·H2O, Fmoc-Lys(ivDde)-OH, Fmoc-Ala-OH·H2O, Fmoc-Lys(Boc)-OH, It includes sequential coupling steps of Fmoc-Lys(Boc)-OH, Fmoc-L-Glu(OtBu)-OH·H2O, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Gln(Trt)-OH, Fmoc-Aib-OH, and Boc-His(Trt)-OH.

[0111] In some embodiments, solid-phase synthesis is performed on a Fmoc amide resin solid support and includes a sequential coupling step of Fmoc amino acids of the following Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, and Fmoc-Pro-OH·H2O prior to the Fmoc deprotection step of the amide resin and the coupling step of Fmoc-Gly-Gly-OH. After the coupling step of Fmoc-Gly-Gly-OH, the solid-phase synthesis is as follows: Fmoc-L-Glu(OtBu)-OH·H2O, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-L-Glu(OtBu)-OH·H2O, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-L-Glu(OtBu)-OH·H2O, Fmoc-Lys(ivDde)-OH, Fmoc-Ala-OH·H2O, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-L-Glu(OtBu)-OH·H2O, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, It further includes sequential coupling steps of Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Gln(Trt)-OH, Fmoc-Aib-OH, and Boc-His(Trt)-OH.

[0112] In a preferred embodiment, naturally occurring amino acids are preferably used in this application.

[0113] SPPS comprises several basic steps that are repeated as the peptide chain is extended by adding additional amino acids to the elongating peptide chain. The 'solid phase' refers to resin particles (e.g., link-amide resin) to which the elongating peptide chain is attached following the original amino acid. Since the chain is attached to the particles, they can be treated as a set of solid particles (particularly in washing and separation steps, e.g., in the filtration step).

[0114] The repeated steps of SPPS include deprotection, activation, and coupling steps.

[0115] (1) Deprotection step: The final acid on the peptide chain is maintained in a 'protected' state before the start of each cycle. As used herein, the term 'protected' means that a protecting group is attached at a specific location, that is, the 'amino' terminus of the peptide chain is attached to a functional group that protects the acid from unwanted reactions. Various protecting groups are well known, and alternative protecting groups may be suitable for a specific method. When the next amino acid is added, the 'protecting group' is removed ('deprotection' step).

[0116] (2) Activation step: A compound ('activator') is added to the reaction to produce intermediate amino acid species that are more likely to couple with acid after deprotection on the peptide chain.

[0117] (3) Coupling step: The species after activation is coupled to the existing peptide chain.

[0118] The reagent for deprotection may be one commonly used in the field. In the present invention, it is preferable to use a Pip / DMF / NMP solution or a DBU deprotection solution, wherein Pip may be 15% to 20% by weight, and the mass ratio of DMF and NMP is preferably 1: (0.9 to 1.1). For example, preferably, the mass ratio of DMF, Pip, and NMP is 2.2:1:2.4.

[0119] In a preferred embodiment, the deprotection reaction in the solid-phase synthesis of the present invention comprises the step of performing deprotection twice for 13 to 17 minutes (e.g., 14 to 16 minutes or 15 minutes) each in the first 10 solid-phase synthesis cycles, and the step of performing deprotection twice for 25 to 35 minutes (e.g., 29 to 31 minutes or 30 minutes) each in the last 13 to 33 solid-phase synthesis cycles. Reducing the time and frequency of deprotection can shorten the production period and reduce material costs.

[0120] In some embodiments, deprotection may be performed three times (for 15 minutes each) for nucleotide Val, and deprotection may also be performed three times (for 30 minutes each) for amino acid Phe at position 22 after Val. In some embodiments, deprotection may be performed four times (for 30 minutes each) for amino acids Thr at positions 5 and 7.

[0121] One of the most commonly used and studied activation methods for peptide synthesis is in carbodiimide-based applications. Carbodiimide contains two weakly basic nitrogen atoms, which react with the carboxylic acid of an amino acid derivative to form a highly reactive O-acylisourea compound. In a preferred embodiment, 1-hydroxybenzotriazole (HOBt) or 1-hydroxy-7-azabenzotriazole (HOAt) is introduced as an additive to the carbodiimide activation method to significantly improve the carbodiimide activation method. Instead of HOBt or HOAt, other additives, for example, 2-cyano-2-(hydroxyimino)ethyl acetate (oxyma) or 1-hydroxy-2,5-pyrrolidindione (NHS) may be used.

[0122] In addition, onium salt condensation reagents can be used for activation, which are highly reactive and fast. For example, 1H-Benzotriazole-1-yloxytripyrrolidine yl hexafluorophosphate (PyBOP), etc., may be used. For the use of reagents, diisopropylethylamine (DIEA) and to activate amino acids, and N - The addition of an organic base such as methylmorpholine (NMM) is required. 1-hydroxybenzotriazole (HOBt) can also be introduced as an additive in the onium salt condensation reaction.

[0123] Activation can also be performed using an organophosphorus phosphate condensing agent, for example, 3-(diethoxyphosphooxy)-1,2,3-benzotriazine-4-one (DEPBT). DEPBT is an organophosphorus phosphate condensing agent derived from HOBt and has advantages such as good solubility, mild reaction conditions, no need to protect partial amino acid side chains, and a low racemization rate. An organic base, for example, DIEA, can be added to the reaction of the reagent.

[0124] In the method of the present invention, a preferred activation reaction system is DIC / HOBt / HOAt / Oxyma in DMF or DMF / NMP, or PyBOP / HOBt in DMF or DMF / NMP, or DEPBT in DMF or DMF / NMP.

[0125] In one embodiment of the present invention, the molar amount of the activator may be 3 to 7 times greater than the molar amount of the resin. Additionally, the ratio of the molar amount of the additive to the molar amount of the activator may be about 1. If the addition of an organic base is required, the amount of the organic base used is preferably the sum of the molar amounts of the activator and any additive.

[0126] In a preferred embodiment, the coupling step of the first 11 amino acids (i.e., the first 10 solid-phase synthesis cycles) is performed using a low equivalent amount (e.g., 2 to 4 equivalents, preferably 2, 3, or 4 equivalents) of Fmoc amino acids with respect to the resin, and the coupling step of the remaining amino acids is performed using a high equivalent amount (e.g., 4 to 6 equivalents, e.g., 4, 5, or 6 equivalents) of Fmoc amino acids with respect to the resin. In some embodiments, the coupling step at His and Lys at position 20 may be performed using 3 equivalents of Fmoc amino acids.

[0127] In some embodiments, the coupling step may be performed at a temperature of 20 to 30°C for about 3 hours, for example, 2 hours 50 minutes to 3 hours 10 minutes. The reaction time may be adjusted according to the degree of reaction completion, but the total reaction time must be ≤12 hours.

[0128] More generally, in relation to the method of the present invention, the SPPS structure is preferably achieved by a standard Fmoc peptide chemistry technique using sequential coupling with an automated peptide synthesizer. The preferred resin is a link amide resin. DMF / NMP is a preferred solvent system, and DMF is used for swelling of the resin. A Pip / DMF / NMP solution is preferably used for deprotection of the resin. A Pip / DMF / NMP solution or a DBU deprotection solution is preferably used for subsequent Fmoc deprotection treatment, which is treated for 2 x 15 minutes during the first 10 solid-phase synthesis cycles, preferably for 2 x 30 minutes during the subsequent coupling step of amino acids, and may also be treated for 2 x 30 minutes in the case of a difficult coupling step. After deprotection, the resin is preferably washed for 8 x 1 to 3 minutes with 7 to 9 (preferably 8) times the amount of DMF. The amino acid activation step is preferably performed at room temperature (e.g., 20–30 °C) or 0–10 °C for 30 minutes using an activator / DMF / NMP solution optionally containing an additive or an inorganic base. The activation step is performed for 1–6 minutes under stirring. For each individual amino acid, a coupling step of the activated amino acid and the peptide bound to the resin is performed for a specific time. After each coupling step, 7–9 (preferably 8) times the amount of DMF washing solvent is preferably performed for 8 × 1–3 minutes.

[0129] Other cleaning agents, such as DCM and NMP, may also be used for the addition of Val, and the washing step may be performed as many times as necessary.

[0130] The difficult coupling step can also be performed by re-application, for example, by repeating the activation, coupling, and washing steps after washing, or by using a different activator during re-application. Solid-phase synthesis optionally further includes a terminal capping step after the coupling reaction to block unreacted amino groups or acetylate the N-terminus of the intact peptide.

[0131] Preferably, when R2 is ivDde, Lys at position 20 is selectively deprotected by contacting the compound with a solution containing hydrazine hydrate. Also, preferably, the solution contains 1% to 15% by weight of hydrazine hydrate in DMF, NMP, NBP, or DMSO.

[0132] More preferably, the solution contains 8% to 9% by weight of hydrazine hydrate in DMF, for example, about 8.6% by weight.

[0133] tBuO-C obtained through DIC / HOSu in step (ii) 20-γGlu(tBu)-AEEA-AEEA-OSu active ester is directly coupled to the ε-amino group of the Lys side chain at position 20 of the resin peptide obtained in step (1). The side chain is directly coupled to the polypeptide via DIC / HOSu, which shortens the reaction time and reduces the formation of possible racemization and deletion impurities. In step (ii), the molar ratio of DIC / HOSu / side chain may be 1:1:1. HOSu may be slightly in excess. After reacting the side chain with DIC / HOSu in step (ii), the reaction system is filtered and concentrated, dissolved in DCM, filtered, then DIEA and DMF are added, and coupling is performed at room temperature for about 12 hours. After the coupling step, the reaction system is washed 6 times with 8 to 10 times the amount of NMP, then 8 to 10 times (e.g., 8 or 9 times) the amount of MTBE is added, the reaction system is stirred for 1 to 3 minutes and filtered (this operation is performed twice). 8 to 10 times (e.g., 8 or 9 times) the amount of DCM is added, the reaction system is stirred for 1 to 3 minutes and filtered (this operation is performed twice). 8 to 10 times (e.g., 8 or 9 times) the amount of MTBE is added, the reaction system is stirred for 1 to 3 minutes and filtered (this operation is performed twice), and then a drying step is performed.

[0134] For the next step, after ivDde is removed, the reaction system is filtered and sequentially washed with 8 times the amount of DMF (10 times) and NMP (2 times) for 1 to 3 minutes each, and the obtained product is immersed in NMP.

[0135] (iii) Due to the low stability of the crude peptide in the cleavage reaction system in step (iii), it is preferable to use the mixture with the resin directly in the next step after the compound is cleaved on the solid support. That is, after the reaction is completed, the resin is not filtered, and the reaction system is added directly to MTBE for crystallization, and the resin is dissolved and filtered in subsequent treatment.

[0136] Specifically, in step (iii), the peptide in the resin is preferably cleaved with an acidic mixture consisting of trifluoroacetic acid, triisopropylsilane, dithiothreitol, and water. For example, the cleaving reaction system may be TFA:H2O:TIPS:DTT=92.5:4.4:3.1:3 (V / V; DTT w / v).

[0137] (iii) After step, for crystallization, the reaction system after cutting was directly added to MTBE, filtered, dried and ground, slurried with MTBE, and then dried.

[0138] In one embodiment of the method of the present invention, the purification step of the compound includes the step of purifying the solution of the compound of step (iii) through chromatographic purification.

[0139] Preferably, chromatographic purification is performed by HPLC or reverse-phase HPLC.

[0140] More preferably, the purification step further comprises (a) obtaining a product by elution using an excess of ammonium acetate / acetonitrile (ACN) / water mobile phase, performing salt substitution and removing trifluoroacetic acid (TFA), (b) performing concentration and removing the solvent, and (c) performing freeze-drying.

[0141] The present invention provides a method for preparing a compound of formula I (sequence number 3) or a pharmaceutically acceptable salt thereof, and

[0142]

[0143] Chemical Formula I

[0144] The method is

[0145] (i) solid-phase synthesis of a compound of the following chemical formula, wherein

[0146]

[0147] Fmoc-Gly-Gly-OH is used in the corresponding solid-phase synthesis.

[0148] The present invention provides a method for preparing a compound of Formula II (Sequence No. 4) or a pharmaceutically acceptable salt thereof, and

[0149]

[0150] Chemical Formula II

[0151] The method is

[0152] (i) solid-phase synthesis of a compound of the following chemical formula, wherein

[0153]

[0154] Fmoc-Gly-Gly-OH is used in the corresponding solid-phase synthesis,

[0155] (ii) Selective deprotection of Lys is performed at position 20.

[0156] The present invention provides a method for preparing a compound of Formula III (Sequence No. 5) or a pharmaceutically acceptable salt thereof, and

[0157]

[0158] Chemical Formula III

[0159] The method is

[0160] (i) solid-phase synthesis of a compound of the following chemical formula, wherein

[0161]

[0162] Fmoc-Gly-Gly-OH is used in the corresponding solid-phase synthesis,

[0163] (ii) tBuO-C obtained through a selective deprotection step of Lys at position 20 and DIC / HOSu 20 It includes the step of directly coupling the -γGlu(tBu)-AEEA-AEEA-OSu active ester to the ε-amino group of the Lys side chain at position 20.

[0164] In another aspect, the present application provides a compound of the following formula I (SEQ ID NO. 3) or a pharmaceutically acceptable salt thereof, a compound of the following formula II (SEQ ID NO. 4) or a pharmaceutically acceptable salt thereof, or a compound of the following formula III (SEQ ID NO. 5) or a pharmaceutically acceptable salt thereof:

[0165]

[0166] Chemical formula I,

[0167]

[0168] Chemical formula II,

[0169]

[0170] Chemical formula III.

[0171] In some embodiments, the compound of Formula I or its pharmaceutically acceptable salt, the compound of Formula II or its pharmaceutically acceptable salt, or the compound of Formula III or its pharmaceutically acceptable salt is a compound or its pharmaceutically acceptable salt selected from the following compounds or their pharmaceutically acceptable salts:

[0172]

[0173] Compound 2 (Sequence No. 6),

[0174]

[0175] Compound 3 (Sequence No. 7),

[0176]

[0177] Compound 4 (Sequence No. 8).

[0178] definition

[0179] The following abbreviations used herein have the meanings described herein. 'SPPS' indicates solid phase peptide synthesis, 'Fmoc' indicates fluorenylmethoxycarbonyl chloride, 'Boc' indicates tertiary-butoxycarbonyl, 'OtBu' indicates tertiary-butyl ester, 'tBu' indicates tertiary-butyl, 'Trt' indicates triphenylmethyl or trityl, and NMP is N -Methylpyrrolidone( N -methylpyrrolidone) represents, 'ivDde' represents 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl[1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl], 'Pip' represents piperidine, and 'DIC' is N,N -Diisopropylcarbodiimide( N,N '-diisopropylcarbodiimide) represents, 'Oxyma' represents ethyl 2-oxime cyanoacetate, 'DCM' represents dichloromethane, DEPBT represents 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4-one, and DMF is N,N -Dimethylformamide ( N,N-dimethylformamide) represents, 'TFA' represents trifluoroacetic acid, 'TIPS' represents triisopropylsilane, 'DTT' represents dithiothreitol, 'AEEA' represents 17-amino-10-oxo-3,6,12,15-tetraoxa-9-azaheptadecanoic acid, 'HOBt' represents 1-hydroxybenzotriazole, and 'PyBOP' is 1H - Represents benzotriazole-1-yloxytripyrrolidinyl hexafluorophosphate. 'tBuO-C 20 -γ-Glu(tBu)-AEEA-AEEA-OH' represents (S)-22(tert-butoxycarbonyl)-45,45-dimethyl-10,19,24,43-tetraoxo-3,6,12,15,44-pentaoxa-9,18,23-triaz-tetraacetic acid, 'AEEA' represents 8-amino-3,6-dioxaoctanoic acid, HOAt represents 1-hydroxy-7-azabenzotriazole, and DIEA is N,N - Represents diisopropylethylamine.

[0180] As indicated herein, single-letter amino acid abbreviations are shown in bold, and atoms are shown in non-bold and typically have a smaller font size to distinguish them from the single-letter amino acid abbreviations. As used herein, amino acids labeled by abbreviations are accompanied by a number above them indicating the corresponding position of the amino acid in the final mazdutide product. These numbers are provided for convenience, and the presence or absence of these numbers in the sequence does not affect the amino acid sequence or peptide indicated in such sequence. As used herein, the term "protected" means that a protecting group is attached to the indicated position. A person skilled in the art will recognize that various protecting groups are well known and that alternative protecting groups may be suitable for a particular method.

[0181] The amino acid sequence of the present invention includes standard one-letter or three-letter codes for 20 naturally occurring amino acids. Additionally, 'Aib' is α-aminoisobutyric acid.

[0182] The present invention relates to novel compounds and methods that can be used in the synthesis of the compounds disclosed herein or pharmaceutically acceptable salts thereof (particularly acetates). Novel methods and compounds are exemplified in the following examples. Reagents and starting materials are readily available to a person skilled in the art. It will be understood that these examples are not intended to limit the scope of the invention in any way.

[0183] Examples

[0184] All reagents used in this application are commercially available and can be used without further purification.

[0185] Example 1. Synthesis of a compound

[0186] Synthesis path of the side chain

[0187]

[0188] Step 1A: Synthesis of Compound a

[0189] In a 200 L enamel reactor (8.10ХW 1A ) kg of acetonitrile was added, and the reaction system temperature was adjusted to 15–30 ℃. W 1A kg of mono(1,1-dimethylethyl) docosanedioate and (0.03 ХW 1A ) kg of 4-dimethylaminopyridine was added while stirring, (0.77 ХW 1A ) kg of N,N Disuccinimidyl carbonate was added in three separate additions at intervals of 5 to 10 minutes. The funnel ((0.20~0.40)ХW 1A It was washed with ) kg of acetonitrile. The reaction system in the reactor was heated to 35–45 ℃ (target temperature: 40 ℃) and the reaction was carried out for at least 3 hours while maintaining the temperature.

[0190] The reaction system in the reactor was cooled to -5–5 °C, stirred for 2–3 hours, and then filtered. The reactor (1.54 ХW 1A ) kg of acetonitrile, (0.80ХW 1A ) kg of acetonitrile / (0.26ХW 1A ) kg of purified water and (1.00ХW 1A The filter cake was washed sequentially with ) kg of acetonitrile, cooled to -5 to 5 ℃, and pre-cooled for at least 1 hour. The filter cake was washed by immersing it in the washing solution for 5 to 10 minutes, and then filtered while controlling the temperature to ≤5 ℃. The filter cake was dried at 15 to 30 ℃ to obtain compound a, which was then sealed and stored at 10 to 30 ℃.

[0191] W 1A It was 3.39 kg.

[0192] Step 1B: Synthesis of Compound b

[0193] In a 100 L enamel reactor (7.86 GHz) 1B) kg of acetonitrile was added, and the reaction system temperature was adjusted to 15–30 ℃. W 1B kg of compound a and (0.48 ХW 1B ) kg of H-Glu-OtBu was added. The reactor was ((1.00~2.00)ХW 1B It was washed with ) kg of acetonitrile. While controlling the temperature to 15–30 ℃ (0.31 ХW 1B ) kg of N,N - Diisopropylethylamine was added to the reactor. The reaction system in the reactor was heated to 35–45 ℃ (target temperature: 40 ℃) and the reaction was carried out for at least 8 hours while maintaining the temperature.

[0194] While controlling the temperature to 35~45 ℃, in the reactor (0.150ХW 1B ) Acetic acid and (4.00ХW 1B ) kg of purified water was added. The reaction system in the reactor was cooled to 10–15 ℃ while maintaining the temperature for 1–2 hours, then heated to 20–25 ℃ while maintaining the temperature for 1–2 hours. The reaction system was cooled to -5–5 ℃ while maintaining the temperature for 1–3 hours, then filtered while controlling the temperature to -5–5 ℃. The reactor (1.65ХW 1B ) kg of acetonitrile / (0.90ХW 1B ) kg of purified water and (1.57 ХW 1B The filter cake was washed sequentially with ) kg of acetonitrile, cooled to -5 to 5 ℃, and pre-cooled for at least 0.5 hours. The filter cake was washed by immersing it in the washing solution for 5 to 10 minutes and then filtered (the temperature was controlled to ≤5 ℃). The filter cake was dried at 15 to 30 ℃ to obtain compound b, which was then sealed and stored at 10 to 30 ℃.

[0195] W 1B It was 3.85 kg.

[0196] Step 1C: Synthesis of Compound C

[0197] In a 100 L enamel reactor (8.86 GHz) 1C ) kg of acetonitrile was added, and the reaction system was stirred while controlling the reaction system temperature to 15–30 ℃. W 1C kg of compound b and (0.0200ХW 1C ) kg of 4-dimethylaminopyridine was added while stirring, (0.53 ХW 1C ) kg of N,N Disuccinimidyl carbonate was added in three separate additions at intervals of 5 to 10 minutes. The funnel and reactor walls were ((1.02–2.04)ХW 1C It was washed with ) kg of acetonitrile. The reaction system in the reactor was heated to 15–25 ℃ (target temperature: 20 ℃) ​​and the reaction was carried out for at least 3 hours while maintaining the temperature.

[0198] The reaction system in the reactor was cooled to -5 to 5 °C and stirred for at least 2 hours. The reaction system was filtered while controlling the temperature to -5 to 5 °C. The reactor (4.40ХW 1C The filter cake was washed with ) kg of acetonitrile, cooled to -5 to 5 ℃, and pre-cooled for at least 0.5 hours. The filter cake was washed by immersing it in the washing solution a total of 4 times for 5 to 10 minutes each, and then filtered (the temperature was controlled to ≤5 ℃). The filter cake was dried at 15 to 30 ℃ to obtain compound c, which was then sealed and stored at 10 to 30 ℃.

[0199] W 1C It was 4.23 kg.

[0200] Step 1D: Synthesis of Compound 1

[0201] Solution preparation: (13.50 GHz) 1D ) kg of purified water and (1.50ХW 1D A solution was prepared with ) kg of sodium chloride and denoted as N1. (17.60ХW 1D ) kg of purified water, (1.86 kHz 1D ) kg of potassium hydrogen sulfate and (2.00ХW 1DA solution was prepared using ) kg of sodium chloride solution, and after repeating the preparation process twice, the solution was denoted as N2. (16.00ХW 1D ) kg of purified water, (1.60ХW 1D ) kg of potassium hydrogen sulfate and (1.60ХW 1D A solution was prepared with ) kg of sodium chloride solution and labeled as N3.

[0202] In 500 L enamel reactor No. 2 (40.00ХW 1D ) kg of dichloromethane was added, and the reaction system was stirred. (0.60 ХW 1D ) kg of N,N -Diisopropylethylamine and (0.50ХW 1D ) kg of 17-amino-10-oxo-3,6,12,15-tetraoxa-9-azaheptadecanic acid (purchased from Chengdu Pukang Biotechnology Co., Ltd.) was added. The reaction system temperature was adjusted to 15–25 ℃, and the reaction system was stirred for 2–4 hours. W 1D kg of compound c was added to temperature-controlled reactor No. 2, and the funnel and reactor walls (3.00ХW 1D It was washed with ) kg of dichloromethane. The reaction system was reacted at 15–25 ℃ for 8 hours while maintaining the temperature.

[0203] The reaction system in Reactor No. 2 was concentrated at a temperature T ≤30 ℃ and a reference P ≤-0.06 MPa until the residual volume reached 5–10 vol. (10.00 ХW 1D ) kg of ethyl acetate was added to reactor No. 2, and the reaction system was continuously concentrated until the residual volume was 5 to 10 vol. (This process was repeated 3 times).

[0204] In reactor No. 2 (10.00ХW 1D ) kg of ethyl acetate was added, and at a temperature T≤30 ℃, (9.50 THW) was added to Reactor No. 2. 1D ) kg of N3 solution and (5.00ХW1D ) kg of N2 solution was added. The reaction system was stirred for 15–20 minutes and then allowed to stand for liquid separation. N2 solution was added to the organic phase 5 times for washing while controlling the temperature to 15–30 ℃, and at each time (5.00 kHz) 1D ) kg was added, and the mixture was stirred for 15–20 minutes during each wash. Subsequently, the reaction system was allowed to stand for liquid separation. While controlling the temperature to 15–30 ℃, N1 solution was added to reactor No. 2 twice for washing, and (5.00ХW per wash 1D ) kg was added, and the mixture was stirred for 5 to 10 minutes during each wash. Subsequently, the reaction system was left to stand for liquid separation.

[0205] Transfer the organic phase to 3000 L enamel reactor No. 1, and reactor No. 2 ((1.00~2.00)ХW 1D After washing with ) kg of preparative acetonitrile, the washing solution was combined and added to Reactor 1. The reaction system in Reactor 1 was concentrated until the residual volume reached 2–3 vol. at a jacket temperature T ≤30 ℃ and a reference P ≤-0.08 MPa. (3.50 ХW 1D ) kg of preparative acetonitrile was added to reactor No. 1, and the reaction system was continuously concentrated until the residual volume was 2 to 3 vol. (This process was repeated 3 times).

[0206] (6.00~8.00ХW 1D ) kg of preparative acetonitrile was added to Reactor 1, and stirring was started. The reaction system was cooled to -20 to -10 ℃ (target: -13 ℃) and stirred for 3 to 5 hours while maintaining the temperature. The reaction system in Reactor 1 was filtered at a controlled temperature of -20 to -10 ℃ and pressure-filtered with pre-cooled nitrogen. Reactor 1 was ((1.00~3.00)ХW 1DThe sample was washed with a preparative acetonitrile, and the jacket was pre-cooled for at least 0.5 hours with a cooling control of -5 to 5 °C. The filter cake was immersed in a washing solution, washed twice for 5 to 10 minutes each, and then filtered. The reaction system was dried at -15 °C to -5 °C to obtain Compound 1, which was then sealed and stored at -25 to -15 °C.

[0207] W 1D It was 4.65 kg.

[0208] Example 2. Preparation of Mazdutide

[0209] Main synthesis path

[0210]

[0211]

[0212] 2.1 Preparation of Compound 2

[0213] Solution preparation

[0214] DMF / NMP mixed solution: N,N - Dimethylformamide and N Methylpyrrolidone was mixed in a weight ratio of 1:1.09, and the mixture was stirred for 30 to 60 minutes to prepare the mixed solution.

[0215] Pip / DMF / NMP Solution: N,N - Dimethylformamide, piperidine, N Methylpyrrolidone (at a weight ratio of 2.2:1:2.4) was added in batches, and pumped and circulated with a magnetic pump for 2 to 4 hours to prepare a mixed solution.

[0216] DBU deprotection solution: piperidine, 1,8-diazabicyclodec-7-en, N,N - Dimethylformamide and N - Methylpyrrolidone was added in a weight ratio of 1:1.2:26.3:28.6 and then stirred for 30 to 60 minutes to prepare a mixed solution. The solution can be prepared in batches.

[0217] Step 1-1b

[0218] Resin Pretreatment: Link-amide resin (6.82 kg W1, 2.99 mol N) was added to a 500 L synthesis reactor, and to wash the resin twice N,N - Dimethylformamide was added twice (used in each instance N,N - The amount of dimethylformamide was 8 W1, and the reaction system was purged with nitrogen for 3 minutes, followed by stirring for 1 to 3 minutes per cycle. After washing was complete, the reaction system was filtered until no more droplets fell. N,N - Dimethylformamide was removed.

[0219] In a 500 L synthesis reactor N,N - After swelling the resin by adding dimethylformamide (used N,N The amount of dimethylformamide was 8 W1), the reaction system was stirred while purging with nitrogen, and the temperature was controlled to 25±5 ℃. After swelling the resin for 0.5 to 1 hour, the reaction system was filtered until no more droplets fell. N,N - Dimethylformamide was removed.

[0220] Steps 1-2a and 1-2b through 1-34a

[0221] Compound 2 was obtained through the following cycles 1 to 33.

[0222] Fmoc-Gly-OH bond (cycle 1)

[0223] 1) Deprotection: 1 L of 8.0 W 1 ± 1.0 W Pip / DMF / NMP solution was added to a 500 L synthesis reactor via a buffer tank and purged with nitrogen for 3 minutes. Afterward, the reaction system was stirred at a temperature controlled to 20–30 °C. After stirring for 14–16 minutes, stirring was stopped and the nitrogen supply was cut off. The reaction system was filtered to remove the solution until no more droplets fell. This process was repeated twice.

[0224] 2) Washing: 8.0 W 1±1.0 W 1L to wash the resin N,N - Dimethylformamide was added to a 500 L synthesis reactor, and the reaction system was purged with nitrogen for 3 minutes, followed by stirring for 1 to 3 minutes per cycle. After washing was complete, the reaction system was filtered until no more droplets fell. N,N - Dimethylformamide was removed. This process was performed 8 times.

[0225] 3) Activation: (4 W 1X0.986) kg of DMF / NMP solution, (3 N X0.297) kg of Fmoc-Gly-OH, and (3.3 N X0.135) kg of 1-hydroxybenzotriazole were added to an 80 L glass reactor, and (3.3 N X0.126) kg of N,N Diisopropylcarbodiimide was added dropwise at a temperature controlled to 20–30 ℃, and the reaction system was stirred for 4–6 minutes.

[0226] 4) Coupling: After activating the glass reactor, the reaction system was added to a 500 L synthesis reactor, and the reaction system was reacted at a temperature controlled to 20–30 ℃, during which time the walls of the synthesis reactor N,N - It could be washed with dimethylformamide (total amount ≤1.5 NL). After reacting for 1 hour 50 minutes to 2 hours 10 minutes, the reaction system was sampled for ninhydrin detection, and if the resin was colorless, the reaction was continued for 2 hours 50 minutes to 3 hours 10 minutes, and the system was filtered.

[0227] 5) Washing: 8.0 W 1±1.0 W 1L N,N - Dimethylformamide was added to a 500 L synthesis reactor, and the reaction system was purged with nitrogen for 3 minutes, followed by stirring for 1 to 3 minutes per cycle. After washing was complete, the reaction system was filtered until no more droplets fell. N,N - Dimethylformamide was removed. This process was performed 6 times.

[0228] Fmoc-Ser(tBu)-OH bond (cycle 2)

[0229] 1) Deprotection: This task was performed in the same manner as Cycle 1.

[0230] 2) Cleaning: This task was performed in the same manner as Cycle 1.

[0231] 3) Activation: (4 W 1X0.986) kg of DMF / NMP solution, (3 N X0.383) kg of Fmoc-Ser(tBu)-OH, and (3.3 N X0.135) kg of 1-hydroxybenzotriazole were added to an 80 L glass reactor, the reaction system was cooled to 0–10 °C, and (3.3 N X0.126) kg of N,N Diisopropylcarbodiimide was added dropwise at a controlled temperature, and the reaction system was stirred for 4 to 6 minutes.

[0232] 4) Coupling: This task was performed in the same manner as Cycle 1.

[0233] 5) Washing: This task was performed in the same manner as Cycle 1.

[0234] Fmoc-Ser(tBu)-OH bond (cycle 3)

[0235] 1) Deprotection: This task was performed in the same manner as Cycle 1.

[0236] 2) Cleaning: This task was performed in the same manner as Cycle 1.

[0237] 3) Activation: (4 W 1X0.986) kg of DMF / NMP solution, (3 N X0.383) kg of Fmoc-Ser(tBu)-OH, and (3.3 N X0.135) kg of 1-hydroxybenzotriazole were added to an 80 L glass reactor, the reaction system was cooled to 0–10 °C, and (3.3 N X0.126) kg of N,N Diisopropylcarbodiimide was added dropwise at a controlled temperature, and the reaction system was stirred for 4 to 6 minutes.

[0238] 4) Coupling: This task was performed in the same manner as Cycle 1.

[0239] 5) Washing: This task was performed in the same manner as Cycle 1.

[0240] 6) End capping: a) Solution preparation: 4 W 1 x 0.986 kg under stirring N,N - Dimethylformamide and N A mixed solution of -methylpyrrolidone (1:1) was added to an 80 L glass reactor, and after adding 3 N x 0.102 kg of acetic anhydride, 3 N x 0.129 kg of N,N -Diisopropylethylamine( N,N '-diisopropylethylamine (DIPEA) was added. After the drop addition was completed, the reaction system was transferred directly to a 500 L synthesis reactor. b) Terminal capping reaction: The reaction system was stirred while purging with nitrogen, and the temperature was controlled to 20–30 ℃; during this time, the walls were... N,N - It can be washed with dimethylformamide (total amount for washing the walls ≤1.5 NL). After reacting for 55–65 minutes, filter the reaction system until no more droplets fall. N,N - Dimethylformamide was removed. Subsequently, the following steps were performed. c) Washing: 8.0 W 1±1.0 W 1L of N,N - Dimethylformamide was added to a 500 L synthesis reactor, and the reaction system was purged with nitrogen for 3 minutes, followed by stirring for 1 to 3 minutes per cycle. After washing was complete, the reaction system was filtered until no more droplets fell. N,N - Dimethylformamide was removed. This process was performed 6 times.

[0241] Fmoc-Pro-OH·H 2 O(cycle 4)

[0242] 1) Deprotection: This task was performed in the same manner as Cycle 1.

[0243] 2) Cleaning: This task was performed in the same manner as Cycle 1.

[0244] 3) Activation: (4 W 1X0.986) kg of DMF / NMP solution, (3 N X0.353) kg of Fmoc-Pro-OH·H2O, and (3.3 N X0.135) kg of 1-hydroxybenzotriazole were added to an 80 L glass reactor, the reaction system was cooled to 0–10 °C, and (3.3 N X0.126) N,N Diisopropylcarbodiimide was added dropwise at a controlled temperature, and the reaction system was stirred for 4 to 6 minutes.

[0245] 4) Coupling: This task was performed in the same manner as Cycle 1.

[0246] 5) Washing: This task was performed in the same manner as Cycle 1.

[0247] Fmoc-Gly-Gly-OH bond (cycle 5)

[0248] 1) Deprotection: This task was performed in the same manner as Cycle 1.

[0249] 2) Cleaning: This task was performed in the same manner as Cycle 1.

[0250] 3) Activation: (4 W 1X0.986) kg of DMF / NMP solution, (3 N X0.354) kg of Fmoc-Gly-Gly-OH and (3.3 N X0.142) kg of ethyl 2-oxime cyanoacetate were added to an 80 L glass reactor, and (3.3 N X0.126) kg of N,N Diisopropylcarbodiimide was added dropwise at a temperature controlled to 20–30 ℃, and the reaction system was stirred for 4–6 minutes.

[0251] 4) Coupling: This task was performed in the same manner as Cycle 1.

[0252] 5) Washing: This task was performed in the same manner as Cycle 1.

[0253] 6) End capping: This task was performed in the same manner as in Cycle 3.

[0254] Fmoc-L-Glu(OtBu)-OH·H 2 O bond (cycle 6)

[0255] 1) Deprotection: This task was performed in the same manner as Cycle 1.

[0256] 2) Cleaning: This task was performed in the same manner as Cycle 1.

[0257] 3) Activation: (4 W 1X0.986) kg of DMF / NMP solution, (3 N X0.443) kg of Fmoc-L-Glu(OtBu)-OH·H2O, and (3.3 N X0.135) kg of 1-hydroxybenzotriazole were added to an 80 L glass reactor, the reaction system was cooled to 0–10 °C, and (3.3 N X0.126) kg of N,N Diisopropylcarbodiimide was added dropwise at a controlled temperature, and the reaction system was stirred for 4 to 6 minutes.

[0258] 4) Coupling: This task was performed in the same manner as Cycle 1.

[0259] 5) Washing: This task was performed in the same manner as Cycle 1.

[0260] Fmoc-Leu-OH bond (cycle 7)

[0261] 1) Deprotection: This task was performed in the same manner as Cycle 1.

[0262] 2) Cleaning: This task was performed in the same manner as Cycle 1.

[0263] 3) Activation: (4 W 1X0.986) kg of DMF / NMP solution, (3 N X0.353) kg of Fmoc-Leu-OH, and (3.3 N X0.135) kg of 1-hydroxybenzotriazole were added to an 80 L glass reactor, the reaction system was cooled to 0–10 °C, and (3.3 N X0.126) kg of N,N Diisopropylcarbodiimide was added dropwise at a controlled temperature, and the reaction system was stirred for 4 to 6 minutes.

[0264] 4) Coupling: This task was performed in the same manner as Cycle 1.

[0265] 5) Washing: This task was performed in the same manner as Cycle 1.

[0266] Fmoc-Leu-OH bond (cycle 8)

[0267] 1) Deprotection: This task was performed in the same manner as Cycle 1.

[0268] 2) Cleaning: This task was performed in the same manner as Cycle 1.

[0269] 3) Activation: (4 W 1X0.986) kg of DMF / NMP solution, (3 N X0.353) kg of Fmoc-Leu-OH, and (3.3 N X0.135) kg of 1-hydroxybenzotriazole were added to an 80 L glass reactor, the reaction system was cooled to 0–10 °C, and (3.3 N X0.126) kg of N,N Diisopropylcarbodiimide was added dropwise at a controlled temperature, and the reaction system was stirred for 4 to 6 minutes.

[0270] 4) Coupling: This task was performed in the same manner as Cycle 1.

[0271] 5) Washing: This task was performed in the same manner as Cycle 1.

[0272] Fmoc-Trp(Boc)-OH connection (cycle 9)

[0273] 1) Deprotection: This task was performed in the same manner as Cycle 1.

[0274] 2) Cleaning: This task was performed in the same manner as Cycle 1.

[0275] 3) Activation: (4 W 1X0.986) kg of DMF / NMP solution, (3 N X0.527) kg of Fmoc-Trp(Boc)-OH, and (3.3 N X0.135) kg of 1-hydroxybenzotriazole were added to an 80 L glass reactor, the reaction system was cooled to 0–10 °C, and (3.3 N X0.126) kg of N,NDiisopropylcarbodiimide was added dropwise at a controlled temperature, and the reaction system was stirred for 4 to 6 minutes.

[0276] 4) Coupling: This task was performed in the same manner as Cycle 1.

[0277] 5) Washing: This task was performed in the same manner as Cycle 1.

[0278] Fmoc-L-Glu(OtBu)-OH·H 2 O bond (cycle 10)

[0279] 1) Deprotection: This task was performed in the same manner as Cycle 1.

[0280] 2) Cleaning: This task was performed in the same manner as Cycle 1.

[0281] 3) Activation: (4 W 1X0.986) kg of DMF / NMP solution, (3 N X0.443) kg of Fmoc-L-Glu(OtBu)-OH·H2O, and (3.3 N X0.135) kg of 1-hydroxybenzotriazole were added to an 80 L glass reactor, the reaction system was cooled to 0–10 °C, and (3.3 N X0.126) kg of N,N Diisopropylcarbodiimide was added dropwise at a controlled temperature, and the reaction system was stirred for 4 to 6 minutes.

[0282] 4) Coupling: This task was performed in the same manner as Cycle 1.

[0283] 5) Washing: This task was performed in the same manner as Cycle 1.

[0284] Fmoc-Val-OH bond (cycle 11)

[0285] 1) Deprotection: 0.984 kg of (8.0 W1 ± 1.0 W1) x 0.984 kg of DBU deprotection solution was added to a 500 L synthesis reactor. After purging with nitrogen for 3 minutes, the reaction system was stirred while controlling the temperature to 20–30 ℃. After stirring for 14–16 minutes, stirring was stopped and the nitrogen supply was cut off. The reaction system was filtered to remove the solution until no more droplets fell. This process was repeated three times.

[0286] 2) Washing: 8.0 W 1±1.0 W 1L to wash the resin N,N - Dimethylformamide was added to a 500 L synthesis reactor, and the reaction system was purged with nitrogen for 3 minutes, followed by stirring for 1 to 3 minutes per cycle. After washing was complete, the reaction system was filtered until no more droplets fell. N,N -Dimethylformamide was removed. This process was performed twice. The resin was washed six times in the same manner with 8.0 W 1±1.0 W 1L of dichloromethane (DCM). The resin was (8.0 W 1±1.0 W 1)×1.028 Kg of N - It was washed 4 times in the same way with methylpyrrolidone.

[0287] 3) Activation: (4 W 1X0.986) kg of DMF / NMP solution, (5 N X0.339) kg of Fmoc-Leu-OH, and (5.5 N X0.135) kg of 1-hydroxybenzotriazole were added to an 80 L glass reactor, the reaction system was cooled to 0–10 °C, and (5.5 N X0.126) kg of N,N Diisopropylcarbodiimide was added dropwise at a controlled temperature, and the reaction system was stirred for 4 to 6 minutes.

[0288] 4) Coupling: After activating the glass reactor, the reaction system was added to a 500 L synthesis reactor, and the reaction system was reacted at a temperature controlled to 20–30 ℃, during which time the walls of the synthesis reactor N,N- It could be washed with dimethylformamide (total amount ≤1.5 NL). The reaction was carried out for 2 hours 50 minutes to 3 hours 10 minutes and then filtered.

[0289] 5) Washing: 8.0 W 1±1.0 W 1L N,N - Dimethylformamide was added to a 500 L synthesis reactor, and the reaction system was purged with nitrogen for 3 minutes, followed by stirring for 1 to 3 minutes per cycle. After washing was complete, the reaction system was filtered until no more droplets fell. N,N - Dimethylformamide was removed. This process was performed 4 times.

[0290] 6) Fmoc-Val-OH replenishment and binding: a) Activation: (4 W 1X0.986) kg of DMF / NMP solution, (5 N X0.339) kg of Fmoc-Val-OH, and (5.5 N X0.135) kg of 1-hydroxybenzotriazole were added to an 80 L glass reactor, the reaction system was cooled to 0–10 °C, and (5.5 N X0.126) kg of N,N '-Diisopropylcarbodiimide was added dropwise while controlling the temperature, and the reaction system was stirred for 4–6 minutes. b) Coupling: After activating the glass reactor, the reaction system was added to a 500 L synthesis reactor, and the reaction system was reacted at a temperature controlled to 20–30 ℃, during which time the walls of the synthesis reactor N,N - It could be washed with dimethylformamide (total amount ≤1.5 NL). After reacting for 1 hour 50 minutes to 2 hours 10 minutes, the reaction system was sampled for ninhydrin detection; if the resin was colorless, the reaction was continued for 2 hours 50 minutes to 3 hours 10 minutes, and the system was filtered. c) Washing: 8.0 W 1±1.0 W 1L of N,N - Dimethylformamide was added to a 500 L synthesis reactor, and the reaction system was purged with nitrogen for 3 minutes, followed by stirring for 1 to 3 minutes per cycle. After washing was complete, the reaction system was filtered until no more droplets fell. N,N- Dimethylformamide was removed. This process was performed 6 times.

[0291] Fmoc-Phe-OH bond (cycle 12)

[0292] 1) Deprotection: 1 L of 8.0 W 1 ± 1.0 W Pip / DMF / NMP solution was added to a 500 L synthesis reactor through a buffer tank and purged with nitrogen for 3 minutes. Afterward, the reaction system was stirred at a temperature controlled to 20–30 °C. After stirring for 29–31 minutes, stirring was stopped and the nitrogen supply was cut off. The reaction system was filtered to remove the solution until no more droplets fell. This process was repeated three times.

[0293] 2) Washing: 8.0 W 1±1.0 W 1L to wash the resin N,N - Dimethylformamide was added to a 500 L synthesis reactor, and the reaction system was purged with nitrogen for 3 minutes, followed by stirring for 1 to 3 minutes per cycle. After washing was complete, the reaction system was filtered until no more droplets fell. N,N - Dimethylformamide was removed. This process was performed 8 times.

[0294] 3) Activation: (4 W 1X0.986) kg of DMF / NMP solution, (5 N X0.387) kg of Fmoc-Phe-OH, and (5.5 N X0.135) kg of 1-hydroxybenzotriazole were added to an 80 L glass reactor, the reaction system was cooled to 0–10 °C, and (5.5 N X0.126) kg of N,N Diisopropylcarbodiimide was added dropwise at a controlled temperature, and the reaction system was stirred for 4 to 6 minutes.

[0295] 4) Coupling: After activating the glass reactor, the reaction system was added to a 500 L synthesis reactor, and the reaction system was reacted at a temperature controlled to 20–30 ℃, during which time the walls of the synthesis reactor N,N- It could be washed with dimethylformamide (total amount ≤1.5 NL). After reacting for 1 hour 50 minutes to 2 hours 10 minutes, the reaction system was sampled for ninhydrin detection, and if the resin was colorless, the reaction was continued for 2 hours 50 minutes to 3 hours 10 minutes, and the system was filtered.

[0296] 5) Washing: 8.0 W 1±1.0 W 1L N,N - Dimethylformamide was added to a 500 L synthesis reactor, and the reaction system was purged with nitrogen for 3 minutes, followed by stirring for 1 to 3 minutes per cycle. After washing was complete, the reaction system was filtered until no more droplets fell. N,N - Dimethylformamide was removed. This process was performed 6 times.

[0297] Fmoc-L-Glu(OtBu)-OH·H 2 O bond (cycle 13)

[0298] 1) Deprotection: 1 L of 8.0 W 1 ± 1.0 W Pip / DMF / NMP solution was added to a 500 L synthesis reactor through a buffer tank and purged with nitrogen for 3 minutes. Afterward, the reaction system was stirred at a temperature controlled to 20–30 °C. After stirring for 29–31 minutes, stirring was stopped and the nitrogen supply was cut off. The reaction system was filtered to remove the solution until no more droplets fell. This process was repeated twice.

[0299] 2) Washing: 8.0 W 1±1.0 W 1L to wash the resin N,N - Dimethylformamide was added to a 500 L synthesis reactor, and the reaction system was purged with nitrogen for 3 minutes, followed by stirring for 1 to 3 minutes per cycle. After washing was complete, the reaction system was filtered until no more droplets fell. N,N - Dimethylformamide was removed. This process was performed 8 times.

[0300] 3) Activation: (4 W 1X0.986) kg of DMF / NMP solution, (5 N X0.443) kg of Fmoc-L-Glu(OtBu)-OH·H2O, and (5.5 N X0.135) kg of 1-hydroxybenzotriazole were added to an 80 L glass reactor, the reaction system was cooled to 0–10 °C, and (5.5 N X0.126) kg of N,N Diisopropylcarbodiimide was added dropwise at a controlled temperature, and the reaction system was stirred for 4 to 6 minutes.

[0301] 4) Coupling: After activating the glass reactor, the reaction system was added to a 500 L synthesis reactor, and the reaction system was reacted at a temperature controlled to 20–30 ℃, during which time the walls of the synthesis reactor N,N - It could be washed with dimethylformamide (total amount ≤1.5 NL). After reacting for 1 hour 50 minutes to 2 hours 10 minutes, the reaction system was sampled for ninhydrin detection, and if the resin was colorless, the reaction was continued for 2 hours 50 minutes to 3 hours 10 minutes, and the system was filtered.

[0302] 5) Washing: 8.0 W 1±1.0 W 1L N,N - Dimethylformamide was added to a 500 L synthesis reactor, and the reaction system was purged with nitrogen for 3 minutes, followed by stirring for 1 to 3 minutes per cycle. After washing was complete, the reaction system was filtered until no more droplets fell. N,N - Dimethylformamide was removed. This process was performed 6 times.

[0303] Fmoc-Lys(ivDde)-OH coupling reaction (cycle 14)

[0304] 1) Deprotection: This task was performed in the same manner as Cycle 13.

[0305] 2) Cleaning: This task was performed in the same manner as Cycle 13.

[0306] 3) Activation: (4 W 1X0.986) kg of DMF / NMP solution, (3 N X0.575) kg of Fmoc-Lys(ivDde)-OH, and (3.3 N X0.135) kg of 1-hydroxybenzotriazole were added to an 80 L glass reactor, the reaction system was cooled to 0–10 °C, and (3.3 N X0.126) kg of N,N Diisopropylcarbodiimide was added dropwise at a controlled temperature, and the reaction system was stirred for 4 to 6 minutes.

[0307] 4) Coupling: After activating the glass reactor, the reaction system was added to a 500 L synthesis reactor, and the reaction system was reacted at a temperature controlled to 20–30 ℃, during which time the walls of the synthesis reactor N,N - It could be washed with dimethylformamide (total amount ≤1.5 NL). After reacting for 2 hours 50 minutes to 3 hours 10 minutes, the reaction system was sampled for ninhydrin detection, and if the resin was colorless, the reaction was continued for 3 hours 50 minutes to 4 hours 10 minutes, and the system was filtered.

[0308] 5) Washing: This task was performed in the same manner as Cycle 13.

[0309] Fmoc-Ala-OH·H 2 O bond (cycle 15)

[0310] 1) Deprotection: This task was performed in the same manner as Cycle 13.

[0311] 2) Cleaning: This task was performed in the same manner as Cycle 13.

[0312] 3) Activation: (4 W 1X0.986) kg of DMF / NMP solution, (5 N X0.329) kg of Fmoc-Ala-OH·H2O, and (5.5 N X0.135) kg of 1-hydroxybenzotriazole were added to an 80 L glass reactor, the reaction system was cooled to 0–10 °C, and (5.5 N X0.126) kg of N,NDiisopropylcarbodiimide was added dropwise at a controlled temperature, and the reaction system was stirred for 4 to 6 minutes.

[0313] 4) Coupling: This task was performed in the same manner as in Cycle 13.

[0314] 5) Washing: This task was performed in the same manner as Cycle 13.

[0315] 6) End capping: This task was performed in the same manner as in Cycle 3.

[0316] Fmoc-Lys(Boc)-OH bond (cycle 16)

[0317] 1) Deprotection: This task was performed in the same manner as Cycle 13.

[0318] 2) Cleaning: This task was performed in the same manner as Cycle 13.

[0319] 3) Activation: (4 W 1X0.986) kg of DMF / NMP solution, (5 N X0.469) kg of Fmoc-Lys(Boc)-OH, and (5.5 N X0.135) kg of 1-hydroxybenzotriazole were added to an 80 L glass reactor, the reaction system was cooled to 0–10 °C, and (5.5 N X0.126) kg of N,N Diisopropylcarbodiimide was added dropwise at a controlled temperature, and the reaction system was stirred for 4 to 6 minutes.

[0320] 4) Coupling: After activating the glass reactor, the reaction system was added to a 500 L synthesis reactor, and the reaction system was reacted at a temperature controlled to 20–30 ℃, during which time the walls of the synthesis reactor N,N - It could be washed with dimethylformamide (total amount ≤1.5 NL). After reacting for 2 hours 50 minutes to 3 hours 10 minutes, the reaction system was sampled for ninhydrin detection and then filtered.

[0321] 5) Washing: 8.0 W 1±1.0 W 1L N,N- Dimethylformamide was added to a 500 L synthesis reactor, and the reaction system was purged with nitrogen for 3 minutes, followed by stirring for 1 to 3 minutes per cycle. After washing was complete, the reaction system was filtered until no more droplets fell. N,N - Dimethylformamide was removed. This process was performed 4 times.

[0322] 6) Fmoc-Lys(Boc)-OH replenishment: a) Activation: (4 W 1 x 0.986) kg of DMF / NMP solution, (5 N x 0.469) kg of Fmoc-Lys(Boc)-OH and (5 N x 0.520) kg of 1H -Benzotriazole-1-yloxytripyrrolidinyl hexafluorophosphate and (5 N x 0.135) kg of 1-hydroxybenzotriazole were added to an 80 L glass reactor, the reaction system was cooled to 0–10 °C, and (10 N x 0.129) kg of N,N - Diisopropylethylamine was added dropwise while controlling the temperature to 0–15 ℃, and the reaction system was stirred for 1–3 minutes. b) Coupling: After activating the glass reactor, the reaction system was added to a 500 L synthesis reactor, and the reaction system was reacted at a temperature controlled to 20–30 ℃; during this time, the walls of the synthesis reactor N,N - It could be washed with dimethylformamide (total amount ≤1.5 NL). After reacting for 2 hours 50 minutes to 3 hours 10 minutes, the reaction system was sampled for ninhydrin detection, and filtered when the resin was colorless or nearly colorless. c) Washing: 8.0 W 1±1.0 W 1L of N,N - Dimethylformamide was added to a 500 L synthesis reactor, and the reaction system was purged with nitrogen for 3 minutes, followed by stirring for 1 to 3 minutes per cycle. After washing was complete, the reaction system was filtered until no more droplets fell. N,N - Dimethylformamide was removed. This process was performed 6 times.

[0323] Fmoc-Lys(Boc)-OH bond (cycle 17)

[0324] 1) Deprotection: This task was performed in the same manner as Cycle 13.

[0325] 2) Cleaning: This task was performed in the same manner as Cycle 13.

[0326] 3) Activation: (4 W 1X0.986) kg of DMF / NMP solution, (5 N X0.469) kg of Fmoc-Lys(Boc)-OH and (5 N X0.520) kg of 1H -Benzotriazole-1-yloxytripyrrolidinyl hexafluorophosphate and (5.5 N x 0.135) kg of 1-hydroxybenzotriazole were added to an 80 L glass reactor, the reaction system was cooled to 0–10 °C, and (10 N x 0.129) kg of N,N Diisopropylethylamine was added dropwise at a temperature controlled to 0–10 ℃, and the reaction system was stirred for 1–3 minutes.

[0327] 4) Coupling: After activating the glass reactor, the reaction system was added to a 500 L synthesis reactor, and the reaction system was reacted at a temperature controlled to 20–30 ℃, during which time the walls of the synthesis reactor N,N - It could be washed with dimethylformamide (total amount ≤1.5 NL). After reacting for 2 hours 50 minutes to 3 hours 10 minutes, the reaction system was sampled for ninhydrin detection and then filtered.

[0328] 5) Washing: 8.0 W 1±1.0 W 1L N,N - Dimethylformamide was added to a 500 L synthesis reactor, and the reaction system was purged with nitrogen for 3 minutes, followed by stirring for 1 to 3 minutes per cycle. After washing was complete, the reaction system was filtered until no more droplets fell. N,N - Dimethylformamide was removed. This process was performed 4 times.

[0329] 6) Fmoc-Lys(Boc)-OH replenishment: a) Activation: (4 W 1 x 0.986) kg of DMF / NMP solution, (5 N x 0.469) kg of Fmoc-Lys(Boc)-OH and (5 N x 0.520) kg of 1H -Benzotriazole-1-yloxytripyrrolidinyl hexafluorophosphate and (5.5 N x 0.135) kg of 1-hydroxybenzotriazole were added to an 80 L glass reactor, the reaction system was cooled to 0–10 °C, and (10 N x 0.129) kg of N,N - Diisopropylethylamine was added dropwise while controlling the temperature to 0–10 ℃, and the reaction system was stirred for 4–6 minutes. b) Coupling: After activating an 80 L glass reactor, the reaction system was added to a 500 L synthesis reactor, and the reaction system was reacted at a temperature controlled to 20–30 ℃; during this time, the walls of the synthesis reactor N,N - It could be washed with dimethylformamide (total amount ≤1.5 NL). After reacting for 2 hours 50 minutes to 3 hours 10 minutes, the reaction system was sampled for ninhydrin detection, and filtered when the resin was colorless or nearly colorless. c) Washing: 8.0 W 1±1.0 W 1L of N,N - Dimethylformamide was added to a 500 L synthesis reactor, and the reaction system was purged with nitrogen for 3 minutes, followed by stirring for 1 to 3 minutes per cycle. After washing was complete, the reaction system was filtered until no more droplets fell. N,N - Dimethylformamide was removed. This process was performed 6 times.

[0330] Fmoc-L-Glu(OtBu)-OH·H 2 O bond (cycle 18)

[0331] 1) Deprotection: This task was performed in the same manner as Cycle 13.

[0332] 2) Cleaning: This task was performed in the same manner as Cycle 13.

[0333] 3) Activation: (4 W 1X0.986) kg of DMF / NMP solution, (5 N X0.443) kg of Fmoc-L-Glu(OtBu)-OH·H2O and (5 N X0.520) kg of 1H -Benzotriazole-1-yloxytripyrrolidinyl hexafluorophosphate and (5 N x 0.135) kg of 1-hydroxybenzotriazole were added to an 80 L glass reactor, the reaction system was cooled to 0–10 °C, and (10 N x 0.129) kg of N,N Diisopropylethylamine was added dropwise at a temperature controlled to 0–15 ℃, and the reaction system was stirred for 1–3 minutes.

[0334] 4) Coupling: This task was performed in the same manner as in Cycle 13.

[0335] 5) Washing: This task was performed in the same manner as Cycle 13.

[0336] Fmoc-Asp(OtBu)-OH bond (cycle 19)

[0337] 1) Deprotection: This task was performed in the same manner as Cycle 13.

[0338] 2) Cleaning: This task was performed in the same manner as Cycle 13.

[0339] 3) Activation: (4 W 1X0.986) kg of DMF / NMP solution, (5 N X0.411) kg of Fmoc-Asp(OtBu)-OH and (5 N X0.520) kg of 1H -Benzotriazole-1-yloxytripyrrolidinyl hexafluorophosphate and (5 N x 0.135) kg of 1-hydroxybenzotriazole were added to an 80 L glass reactor, the reaction system was cooled to 0–10 °C, and (10 N x 0.129) kg of N,N Diisopropylethylamine was added dropwise at a temperature controlled to 0–15 ℃, and the reaction system was stirred for 1–3 minutes.

[0340] 4) Coupling: This task was performed in the same manner as in Cycle 13.

[0341] 5) Washing: This task was performed in the same manner as Cycle 13.

[0342] Fmoc-Leu-OH bond (cycle 20)

[0343] 1) Deprotection: This task was performed in the same manner as Cycle 13.

[0344] 2) Cleaning: This task was performed in the same manner as Cycle 13.

[0345] 3) Activation: (4 W 1X0.986) kg of DMF / NMP solution, (5 N X0.353) kg of Fmoc-Leu-OH, and (5.5 N X0.135) kg of 1-hydroxybenzotriazole were added to an 80 L glass reactor, the reaction system was cooled to 0–10 °C, and (5.5 N X0.126) kg of N,N Diisopropylcarbodiimide was added dropwise at a controlled temperature, and the reaction system was stirred for 4 to 6 minutes.

[0346] 4) Coupling: This task was performed in the same manner as in Cycle 13.

[0347] 5) Washing: This task was performed in the same manner as Cycle 13.

[0348] Fmoc-Tyr(tBu)-OH bond (cycle 21)

[0349] 1) Deprotection: This task was performed in the same manner as Cycle 13.

[0350] 2) Cleaning: This task was performed in the same manner as Cycle 13.

[0351] 3) Activation: (4 W 1X0.986) kg of DMF / NMP solution, (5 N X0.460) kg of Fmoc-Tyr(tBu)-OH, and (5.5 N X0.135) kg of 1-hydroxybenzotriazole were added to an 80 L glass reactor, the reaction system was cooled to 0–10 °C, and (5.5 N X0.126) kg of N,N Diisopropylcarbodiimide was added dropwise at a controlled temperature, and the reaction system was stirred for 4 to 6 minutes.

[0352] 4) Coupling: This task was performed in the same manner as in Cycle 13.

[0353] 5) Washing: This task was performed in the same manner as Cycle 13.

[0354] Fmoc-Lys(Boc)-OH bond (cycle 22)

[0355] 1) Deprotection: This task was performed in the same manner as Cycle 13.

[0356] 2) Cleaning: This task was performed in the same manner as Cycle 13.

[0357] 3) Activation: (4 W 1X0.986) kg of DMF / NMP solution, (5 N X0.469) kg of Fmoc-Lys(Boc)-OH and (5 N X0.520) kg of 1H -Benzotriazole-1-yloxytripyrroldinyl hexafluorophosphate and (5 N x 0.135) kg of 1-hydroxybenzotriazole were added to an 80 L glass reactor, the reaction system was cooled to 0–10 °C, and (10 N x 0.129) kg of N,N Diisopropylethylamine was added dropwise at a temperature controlled to 0–15 ℃, and the reaction system was stirred for 1–3 minutes.

[0358] 4) Coupling: After activating the glass reactor, the reaction system was added to a 500 L synthesis reactor, and the reaction system was reacted at a temperature controlled to 20–30 ℃, during which time the walls of the synthesis reactor N,N - It could be washed with dimethylformamide (total amount ≤1.5 NL). After reacting for 2 hours 50 minutes to 3 hours 10 minutes, the reaction system was sampled for ninhydrin detection and then filtered.

[0359] 5) Washing: 8.0 W 1±1.0 W 1L N,N- Dimethylformamide was added to a 500 L synthesis reactor, and the reaction system was purged with nitrogen for 3 minutes, followed by stirring for 1 to 3 minutes per cycle. After washing was complete, the reaction system was filtered until no more droplets fell. N,N - Dimethylformamide was removed. This process was performed 4 times.

[0360] 6) Fmoc-Lys(Boc)-OH replenishment: a) Activation: (4 W 1 x 0.986) kg of DMF / NMP solution, (5 N x 0.469) kg of Fmoc-Lys(Boc)-OH and (5 N x 0.520) kg of 1H -Benzotriazole-1-yloxytripyrrolidinyl hexafluorophosphate and (5 N x 0.135) kg of 1-hydroxybenzotriazole were added to an 80 L glass reactor, the reaction system was cooled to 0–10 °C, and (10 N x 0.129) kg of N,N - Diisopropylethylamine was added dropwise at a temperature controlled to 0–10 ℃, and the reaction system was stirred for 4–6 minutes. b) Coupling: After activating the glass reactor, the reaction system was added to a 500 L synthesis reactor, and the reaction system was reacted at a temperature controlled to 20–30 ℃; meanwhile, the walls of the synthesis reactor N,N - It could be washed with dimethylformamide (total amount ≤1.5 NL). After reacting for 2 hours 50 minutes to 3 hours 10 minutes, the reaction system was sampled for ninhydrin detection and then filtered. c) Washing: 8.0 W 1±1.0 W 1L of N,N - Dimethylformamide was added to a 500 L synthesis reactor, and the reaction system was purged with nitrogen for 3 minutes, followed by stirring for 1 to 3 minutes per cycle. After washing was complete, the reaction system was filtered until no more droplets fell. N,N - Dimethylformamide was removed. This process was performed 6 times.

[0361] 7) End capping: This task was performed in the same manner as in Cycle 3.

[0362] Fmoc-Ser(tBu)-OH bond (cycle 23)

[0363] 1) Deprotection: This task was performed in the same manner as Cycle 13.

[0364] 2) Cleaning: This task was performed in the same manner as Cycle 13.

[0365] 3) Activation: (4 W 1X0.986) kg of DMF / NMP solution, (5 N X0.383) kg of Fmoc-Ser(tBu)-OH, and (5.5 N X0.136) kg of 1-hydroxy-7-azabentriazole were added to an 80 L glass reactor, the reaction system was cooled to 0–10 °C, and (5.5 N X0.126) kg of N,N Diisopropylcarbodiimide was added dropwise at a controlled temperature, and the reaction system was stirred for 4 to 6 minutes.

[0366] 4) Coupling: This task was performed in the same manner as in Cycle 13.

[0367] 5) Washing: This task was performed in the same manner as Cycle 13.

[0368] Fmoc-Tyr(tBu)-OH bond (cycle 24)

[0369] 1) Deprotection: This task was performed in the same manner as Cycle 13.

[0370] 2) Cleaning: This task was performed in the same manner as Cycle 13.

[0371] 3) Activation: (4 W 1X0.986) kg of DMF / NMP solution, (5 N X0.460) kg of Fmoc-Tyr(tBu)-OH, and (5.5 N X0.135) kg of 1-hydroxybenzotriazole were added to an 80 L glass reactor, the reaction system was cooled to 0–10 °C, and (5.5 N X0.126) kg of N,N Diisopropylcarbodiimide was added dropwise at a controlled temperature, and the reaction system was stirred for 4 to 6 minutes.

[0372] 4) Coupling: This task was performed in the same manner as in Cycle 13.

[0373] 5) Washing: This task was performed in the same manner as Cycle 13.

[0374] Fmoc-Asp(OtBu)-OH bond (cycle 25)

[0375] 1) Deprotection: This task was performed in the same manner as Cycle 13.

[0376] 2) Cleaning: This task was performed in the same manner as Cycle 13.

[0377] 3) Activation: (4 W 1X0.986) kg of DMF / NMP solution, (5 N X0.411) kg of Fmoc-Asp(OtBu)-OH and (5 N X0.520) kg of 1H -Benzotriazole-1-yloxytripyrrolidinyl hexafluorophosphate and (5 N x 0.135) kg of 1-hydroxybenzotriazole were added to an 80 L glass reactor, the reaction system was cooled to 0–10 °C, and (10 N x 0.129) kg of N,N Diisopropylethylamine was added dropwise at a temperature controlled to 0–15 ℃, and the reaction system was stirred for 1–3 minutes.

[0378] 4) Coupling: This task was performed in the same manner as in Cycle 13.

[0379] 5) Washing: This task was performed in the same manner as Cycle 13.

[0380] Fmoc-Ser(tBu)-OH bond (cycle 26)

[0381] 1) Deprotection: This task was performed in the same manner as Cycle 13.

[0382] 2) Cleaning: This task was performed in the same manner as Cycle 13.

[0383] 3) Activation: (4 W 1X0.986) kg of DMF / NMP solution, (5 N X0.383) kg of Fmoc-Ser(tBu)-OH, and (5.5 N X0.136) kg of 1-hydroxy-7-azabentriazole were added to an 80 L glass reactor, the reaction system was cooled to 0–10 °C, and (5.5 N X0.126) kg of N,N Diisopropylcarbodiimide was added dropwise at a controlled temperature, and the reaction system was stirred for 4 to 6 minutes.

[0384] 4) Coupling: This task was performed in the same manner as in Cycle 13.

[0385] 5) Washing: This task was performed in the same manner as Cycle 13.

[0386] Fmoc-Thr(tBu)-OH bond (cycle 27)

[0387] 1) Deprotection: 1 L of 8.0 W 1 ± 1.0 W Pip / DMF / NMP solution was added to a 500 L synthesis reactor through a buffer tank and purged with nitrogen for 3 minutes. Afterward, the reaction system was stirred at a temperature controlled to 20–30 °C. After stirring for 29–31 minutes, stirring was stopped and the nitrogen supply was cut off. The reaction system was filtered to remove the solution until no more droplets fell. This process was repeated 4 times.

[0388] 2) Cleaning: This task was performed in the same manner as Cycle 13.

[0389] 3) Activation: (4 W 1X0.986) kg of DMF / NMP solution, (5 N X0.398) kg of Fmoc-Thr(tBu)-OH and (5 N X0.520) kg of 1H -Benzotriazole-1-yloxytripyrrolidinyl hexafluorophosphate and (5 N x 0.135) kg of 1-hydroxybenzotriazole were added to an 80 L glass reactor, the reaction system was cooled to 0–10 °C, and (10 N x 0.129) kg of N,NDiisopropylethylamine was added dropwise at a temperature controlled to 0–15 ℃, and the reaction system was stirred for 1–3 minutes.

[0390] 4) Coupling: This task was performed in the same manner as in Cycle 13.

[0391] 5) Washing: This task was performed in the same manner as Cycle 13.

[0392] 6) End capping: This task was performed in the same manner as in Cycle 3.

[0393] Fmoc-Phe-OH bond (cycle 28)

[0394] 1) Deprotection: This task was performed in the same manner as Cycle 13.

[0395] 2) Cleaning: This task was performed in the same manner as Cycle 13.

[0396] 3) Activation: (4 W 1X0.986) kg of DMF / NMP solution, (5 N X0.387) kg of Fmoc-Phe-OH, and (5.5 N X0.136) kg of 1-hydroxy-7-azabentriazole were added to an 80 L glass reactor, the reaction system was cooled to 0–10 °C, and (5.5 N X0.126) kg of N,N Diisopropylcarbodiimide was added dropwise at a controlled temperature, and the reaction system was stirred for 4 to 6 minutes.

[0397] 4) Coupling: This task was performed in the same manner as in Cycle 13.

[0398] 5) Washing: This task was performed in the same manner as Cycle 13.

[0399] Fmoc-Thr(tBu)-OH bond (cycle 29)

[0400] 1) Deprotection: 1 L of 8.0 W 1 ± 1.0 W Pip / DMF / NMP solution was added to a 500 L synthesis reactor through a buffer tank and purged with nitrogen for 3 minutes. Afterward, the reaction system was stirred at a temperature controlled to 20–30 °C. After stirring for 29–31 minutes, stirring was stopped and the nitrogen supply was cut off. The reaction system was filtered to remove the solution until no more droplets fell. This process was repeated 4 times.

[0401] 2) Cleaning: This task was performed in the same manner as Cycle 13.

[0402] 3) Activation: (4 W 1X0.986) kg of DMF / NMP solution, (5 N X0.398) kg of Fmoc-Thr(tBu)-OH and (5 N X0.520) kg of 1H -Benzotriazole-1-yloxytripyrrolidinyl hexafluorophosphate and (5 N x 0.135) kg of 1-hydroxybenzotriazole were added to an 80 L glass reactor, the reaction system was cooled to 0–10 °C, and (10 N x 0.129) kg of N,N Diisopropylethylamine was added dropwise at a temperature controlled to 0–15 ℃, and the reaction system was stirred for 1–3 minutes.

[0403] 4) Coupling: This task was performed in the same manner as in Cycle 13.

[0404] 5) Washing: This task was performed in the same manner as Cycle 13.

[0405] 6) End capping: This task was performed in the same manner as in Cycle 3.

[0406] Fmoc-Gly-OH reaction (cycle 30)

[0407] 1) Deprotection: This task was performed in the same manner as Cycle 13.

[0408] 2) Cleaning: This task was performed in the same manner as Cycle 13.

[0409] 3) Activation: (4 W 1X0.986) kg of DMF / NMP solution, (5 N X0.297) kg of Fmoc-Gly-OH, and (5.5 N X0.135) kg of 1-hydroxybenzotriazole were added to an 80 L glass reactor, and (5.5 N X0.126) kg of N,N Diisopropylcarbodiimide was added dropwise at a temperature controlled to 20–30 ℃, and the reaction system was stirred for 4–6 minutes.

[0410] 4) Coupling: This task was performed in the same manner as in Cycle 13.

[0411] 5) Washing: This task was performed in the same manner as Cycle 13.

[0412] Fmoc-Gln(Trt)-OH bond (cycle 31)

[0413] 1) Deprotection: This task was performed in the same manner as Cycle 13.

[0414] 2) Cleaning: This task was performed in the same manner as Cycle 13.

[0415] 3) Activation: (4 W 1X0.986) kg of DMF / NMP solution, (5 N X0.611) kg of Fmoc-Gln(Trt)-OH and (5 N X0.520) kg of 1H -Benzotriazole-1-yloxytripyrroldinyl hexafluorophosphate and (5 N x 0.135) kg of 1-hydroxybenzotriazole were added to an 80 L glass reactor, the reaction system was cooled to 0–10 °C, and (10 N x 0.129) kg of N,N Diisopropylethylamine was added dropwise at a temperature controlled to 0–15 ℃, and the reaction system was stirred for 1–3 minutes.

[0416] 4) Coupling: After activating the glass reactor, the reaction system was added to a 500 L synthesis reactor, and the reaction system was reacted at a temperature controlled to 20–30 ℃, during which time the walls of the synthesis reactor N,N- It could be washed with dimethylformamide (total amount ≤1.5 NL). After reacting for 2 hours 50 minutes to 3 hours 10 minutes, the reaction system was sampled for ninhydrin detection and then filtered.

[0417] 5) Washing: 8.0 W 1±1.0 W 1L N,N - Dimethylformamide was added to a 500 L synthesis reactor, and the reaction system was purged with nitrogen for 3 minutes, followed by stirring for 1 to 3 minutes per cycle. After washing was complete, the reaction system was filtered until no more droplets fell. N,N - Dimethylformamide was removed. This process was performed 4 times.

[0418] 6) Fmoc-Gln(Trt)-OH replenishment: a) Activation: (4 W 1 x 0.986) kg of DMF / NMP solution, (5 N x 0.611) kg of Fmoc-Gln(Trt)-OH and (5 N x 0.520) kg of 1H -Benzotriazole-1-yloxytripyrrolidinyl hexafluorophosphate and (5 N x 0.135) kg of 1-hydroxybenzotriazole were added to an 80 L glass reactor, the reaction system was cooled to 0–10 °C, and (10 N x 0.129) kg of N,N - Diisopropylethylamine was added dropwise at a temperature controlled to 0–10 ℃, and the reaction system was stirred for 4–6 minutes. b) Coupling: After activating the glass reactor, the reaction system was added to a 500 L synthesis reactor, and the reaction was carried out while controlling the temperature of the reaction system to 20–30 ℃; during this time, the walls of the synthesis reactor N,N - It could be washed with dimethylformamide (total amount ≤1.5 NL). After reacting for 2 hours 50 minutes to 3 hours 10 minutes, the reaction system was sampled for ninhydrin detection and then filtered. c) Washing: 8.0 W 1±1.0 W 1L of N,N- Dimethylformamide was added to a 500 L synthesis reactor, and the reaction system was purged with nitrogen for 3 minutes, followed by stirring for 1 to 3 minutes per cycle. After washing was complete, the reaction system was filtered until no more droplets fell. N,N - Dimethylformamide was removed. This process was performed 6 times.

[0419] 7) End capping: This task was performed in the same manner as in Cycle 3.

[0420] Fmoc-Aib-OH linkage (cycle 32)

[0421] 1) Deprotection: This task was performed in the same manner as Cycle 13.

[0422] 2) Cleaning: This task was performed in the same manner as Cycle 13.

[0423] 3) Activation: (4 W 1 x 0.986) kg of DMF / NMP solution, (5 N x 0.325) kg of Fmoc-Aib-OH and (5 N x 0.520) kg of 1H -Benzotriazole-1-yloxytripyrrolidinyl hexafluorophosphate and (5 N x 0.135) kg of 1-hydroxybenzotriazole were added to an 80 L glass reactor, the reaction system was cooled to 0–10 °C, and (10 N x 0.129) kg of N,N Diisopropylethylamine was added dropwise at a temperature controlled to 0–15 ℃, and the reaction system was stirred for 1–3 minutes.

[0424] 4) Coupling: After activating the glass reactor, the reaction system was added to a 500 L synthesis reactor, and the reaction system was reacted at a temperature controlled to 20–30 ℃, during which time the walls of the synthesis reactor N,N - It could be washed with dimethylformamide (total amount ≤1.5 NL). After reacting for 2 hours 50 minutes to 3 hours 10 minutes, the reaction system was sampled for ninhydrin detection and then filtered.

[0425] 5) Washing: 8.0 W 1±1.0 W 1L N,N- Dimethylformamide was added to a 500 L synthesis reactor, and the reaction system was purged with nitrogen for 3 minutes, followed by stirring for 1 to 3 minutes per cycle. After washing was complete, the reaction system was filtered until no more droplets fell. N,N - Dimethylformamide was removed. This process was performed 4 times.

[0426] 6) Fmoc-Aib-OH replenishment: a) Activation: (4 W 1 x 0.986) kg of DMF / NMP solution, (5 N x 0.325) kg of Fmoc-Aib-OH and (5 N x 0.520) kg of 1H -Benzotriazole-1-yloxytripyrrolidinyl hexafluorophosphate and (5 N x 0.135) kg of 1-hydroxybenzotriazole were added to an 80 L glass reactor, and (10 N x 0.129) kg of N,N - Diisopropylethylamine was added dropwise at a temperature controlled to 20–30 ℃, and the reaction system was stirred for 4–6 minutes. b) Coupling: After activating an 80 L glass reactor, the reaction system was added to a 500 L synthesis reactor, and the reaction was carried out while controlling the temperature of the reaction system to 20–30 ℃; during this time, the walls of the synthesis reactor N,N - It could be washed with dimethylformamide (total amount ≤1.5 NL). After reacting for 2 hours 50 minutes to 3 hours 10 minutes, the reaction system was sampled for ninhydrin detection, and filtered if the resin was colorless. c) Washing: 8.0 W 1±1.0 W 1L of N,N - Dimethylformamide was added to a 500 L synthesis reactor, and the reaction system was purged with nitrogen for 3 minutes, followed by stirring for 1 to 3 minutes per cycle. After washing was complete, the reaction system was filtered until no more droplets fell. N,N - Dimethylformamide was removed. This process was performed 6 times.

[0427] 7) End capping: This task was performed in the same manner as in Cycle 3.

[0428] Boc-His(Trt)-OH coupling reaction (cycle 33)

[0429] 1) Deprotection: This task was performed in the same manner as Cycle 13.

[0430] 2) Cleaning: This task was performed in the same manner as Cycle 13.

[0431] 3) Activation: (4 W 1 x 0.986) kg of DMF / NMP solution, (3 N x 0.498) kg of Boc-L-His(Trt)-OH, and (6 N x 0.299) kg of 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazine-4-one were added to an 80 L glass reactor, the reaction system was cooled to 0–10 °C, and (6 N x 0.129) kg of N,N Diisopropylethylamine was added dropwise at a controlled temperature, and the reaction system was stirred for 1 to 3 minutes.

[0432] 4) Coupling: After activating the glass reactor, the reaction system was added to a 500 L synthesis reactor, and the reaction system was reacted at a temperature controlled to 22–28 ℃, during which time the walls of the synthesis reactor N,N - It could be washed with dimethylformamide (total amount ≤1.5 NL). The reaction was carried out for 2 hours 50 minutes to 3 hours 10 minutes and then filtered.

[0433] 5) Washing: 8.0 W 1±1.0 W 1L N,N - Dimethylformamide was added to a 500 L synthesis reactor, and the reaction system was purged with nitrogen for 3 minutes, followed by stirring for 1 to 3 minutes per cycle. After washing was complete, the reaction system was filtered until no more droplets fell. N,N - Dimethylformamide was removed. This process was performed 4 times.

[0434] 6) Replenishment of Boc-L-His(Trt)-OH: a) Activation: (4 W 1 x 0.986) kg of DMF / NMP solution, (3 N x 0.498) kg of Boc-L-His(Trt)-OH, and (6 N x 0.299) kg of 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazine-4-one were added to an 80 L glass reactor, the reaction system was cooled to 0–10 °C, and (6 N x 0.129) kg of N,N - Diisopropylethylamine was added dropwise at a controlled temperature, and the reaction system was stirred for 1–3 minutes. b) Coupling: After activating the glass reactor, the reaction system was added to a 500 L synthesis reactor, and the reaction system was reacted at a controlled temperature of 22–28 ℃, during which time the walls of the synthesis reactor N,N - It could be washed with dimethylformamide (total amount ≤1.5 NL). The reaction system was reacted for 2 hours 50 minutes to 3 hours 10 minutes and then filtered. c) Washing: 8.0 W 1±1.0 W 1L of N,N - Dimethylformamide was added to a 500 L synthesis reactor, and the reaction system was purged with nitrogen for 3 minutes, followed by stirring for 1 to 3 minutes per cycle. After washing was complete, the reaction system was filtered until no more droplets fell. N,N - Dimethylformamide was removed. This process was performed 6 times.

[0435] 7) End capping: This task was performed in the same manner as in Cycle 3.

[0436] Polypeptide Analysis Method:

[0437] Agilent 1290 UPLC equipped with a UV detector and a temperature-controlled autosampler or equivalent equipment;

[0438] Chromatography Column: ACQUITY UPLC® Peptide CSH TM , 1.7 μm 2.1X150 mm and an analytical balance with a precision of 0.01 mg.

[0439] The analysis was performed according to the following chromatography conditions.

[0440]

[0441] The polypeptides obtained from the cycle were analyzed according to the polypeptide analysis described above. It was confirmed that the risk of Gly impurity formation could be reduced by performing Gly-Gly coupling at positions 29–30 of the peptide chain using the coupling of Fmoc-Gly-Gly-OH (cycle 5) instead of stepwise coupling of Fmoc-Gly-OH. Additionally, by replacing the coupling of the glycine-threonine pseudoproline dipeptide with the stepwise coupling of Gly and Thr at positions 4 and 5 (corresponding to cycles 29 and 30), no peptides with deleted corresponding amino acids were found, and material costs were reduced.

[0442] The aforementioned polypeptide cycle was also used to generate polypeptides in the next step.

[0443] 2.2 Preparation of Compound 3

[0444] Steps 1–35: Compound 3 was obtained by removing the iVDde protecting group of Lys at position 20 from compound 2 in the following manner.

[0445] (16.2 x W x 0.944) kg of N,N ⅓-dimethylformamide and (1.8 x 1 x 1.026) kg of 80% hydrazine hydrate solution were added to an 80 L glass reactor, and the reaction system was stirred for 15–20 minutes to prepare a mixed solution. The prepared mixed solution was added to a synthesis reactor and stirred for 11 hours 50 minutes to 12 hours 10 minutes while purging with nitrogen at a temperature controlled to 22–32 °C, then filtered until no more droplets fell. N,N - Dimethylformamide was removed (this process was performed twice). 9.0 W 1±1.0 W 1L of N,N- Dimethylformamide was added to the reaction synthesizer, purged with nitrogen for 3 minutes, and stirred for 1 to 3 minutes per cycle. After washing was complete, the reaction system was filtered until no droplets fell off. N,N - Dimethylformamide was removed, and this process was performed 10 times. (9±1.0) W1×1.028 Kg N - The reaction system was washed with methylpyrrolidone in the same manner, and this process was performed twice. In this way, Compound 3 was obtained.

[0446] N - Methylpyrrolidone was added to the synthesis reactor and the resin was immersed until the next step.

[0447] 2.3 Preparation of Compound 4

[0448] Steps 1–36: Compound 4 was obtained by coupling Compound 1 and Compound 3 in the following manner.

[0449] 1) Activation: (7.5 W 1X1.325) kg of dichloromethane was added to an 80 L glass reactor, (1.8 N X0.874) kg of compound 1, and (1.89 N X0.115) kg of N -Hydroxysuccinimide (HOSu) and (1.8 N x 0.126) kg of (1.8 N x 0.126) N,N Diisopropylcarbodiimide (DIC) was added at a temperature controlled to 20–30 ℃. The reaction system was maintained at a temperature of 20–30 ℃ and reacted for 5 hours, after which samples were taken once every 2–4 hours until the peak area percentage of Compound 1 became ≤10%.

[0450] 2) Post-treatment: The reaction system was filtered and then transferred to a glass reactor. The external bath temperature T was adjusted to ≤35 ℃ and the reference pressure P to ≤-0.06 MPa, and the mixture was concentrated until the residual volume reached (1~2 x W1) L. (7.5 W1 x 1.325) kg of dichloromethane was added to reactor No. 2. The reaction system was stirred until no viscous liquid aggregation was visually observed, and then filtered. The filtered reaction system was transferred to a glass reactor (the glass was washed with DCM after filtration) and cooled to 5~15 ℃. (3.6 N x 0.129) kg of N,N - Diisopropylethylamine and (3 W 1X 0.944) kg of N,N - Dimethylformamide was added at a controlled temperature to obtain an active ester solution of compound 1.

[0451] 3) Coupling: The reaction system was transferred to a synthesis reactor after activation and post-treatment. The reaction system was reacted at a temperature controlled to 20–30 ℃, and during this time, the walls of the synthesis reactor N,N - It could be washed with dimethylformamide (total amount ≤1.5 NL). After reacting for 11 hours 50 minutes to 12 hours 10 minutes, the reaction system was sampled for Kaiser detection, and the reaction liquid was discharged when the resin was colorless.

[0452] 4) Washing, shrinking, and drying: 1) Washing: The reaction system is 9 W 1±1 W 1L N,N- It was washed 6 times with dimethylformamide. 2) Shrinkage: 0.740 kg of (9 W1±1 W1) x methyl tertiary-butyl ether was added to a synthesis reactor at 25±5 °C, and the reaction system was purged with nitrogen for 3 minutes, followed by stirring for 1–3 minutes per cycle. Stirring was stopped, the nitrogen was cut off, and the reaction system was filtered to remove the methyl tertiary-butyl ether until no more droplets fell (this process was repeated twice). The reaction system was treated in the same manner with 1 L of 9 W1±1 W dichloromethane. This process was repeated twice. The reaction system was treated in the same manner with 0.740 kg of (9 W1±1 W1) x methyl tertiary-butyl ether. This process was repeated twice. 3) Drying: The reaction system was purged with nitrogen for 4–8 hours, then transferred to a vacuum drying oven for drying to obtain Compound 4.

[0453] 2.4 Preparation of Mazdutide Crude Product

[0454] Step 2: The resin of Compound 4 was cleaved and deprotected in the following manner to obtain the crude peptide of mazdutide. The weight of Compound 4 used was 24.76 kg, denoted as W2.

[0455] 1. Pre-cooling of crystallization solvent: (30 x 2 x 0.740) kg of methyl tert-butyl ether was added to a glass reactor, and the reaction system was cooled to -15 to 0°C.

[0456] 2. Peptide resin cleavage: (10 x W2 x 0.925 x 1.489) kg of trifluoroacetic acid, (10 x W2 x 0.773 x 0.031) kg of triisopropylsilane, (0.3 x W2) kg of dithiothreitol, and (9 x W2 x 0.044) kg of purified water were added to a synthesis reactor, and the reaction system was stirred until the solid was completely dissolved. The reaction system was cooled to -10 to 0 ℃, and W2 kg of Compound 4 was added. The reaction system of the synthesis reactor was heated to 18 to 24 ℃, and the reaction was carried out for 110 to 130 minutes (starting from the point when the reaction system was heated to 18 ℃) while maintaining the temperature.

[0457] 3. Crystallization: The temperature of the glass reactor was adjusted to -15 to 20 ℃, and the reaction system of the synthesis reactor was transferred to the glass reactor. The temperature of the reaction system in the glass reactor was adjusted to 0 to 20 ℃, and the reaction system was stirred for 2 to 4 hours, after which it was filtered. The glass reactor was washed with (5 x W 2 x 0.740) kg of methyl tert-butyl ether, and after immersing the filter cake, it was washed with the washing solution for 20 to 40 minutes (10 times).

[0458] 4. Grinding, Slurrying, and Drying: The material was dried at 20–30 °C to loosen it, recovered, and the solid was ground. (10 x W 2 x 0.740) kg of methyl tert-butyl ether was added to the solid at a temperature of 10 °C, and the reaction system was stirred for 4–8 hours and then filtered. The reaction system was washed with (5 x W 2 x 0.740) kg of methyl tert-butyl ether, the filter cake was immersed, washed in the washing solution for 20–40 minutes, and then filtered (3 times). The material was dried at a temperature of 20–30 °C until the LOD was ≤5.0 % to obtain the crude product of mazdutide (purity: 69%–74%).

[0459] 2.5 Isolation and Purification of Mazdutide

[0460] Step 3: The crude product of mazdutide prepared in Step 2.4 was purified by chromatography and salt transfer methods and then freeze-dried.

[0461] Chromatography

[0462] 3.23 kg of crude mazdutide product was dissolved in acetonitrile / water (ratio of 1 / 1.3), and the reaction system was stirred for 1 hour to 1 hour 30 minutes. After checking the pH value (pH=2~4, the reaction system can be adjusted with 3% diluted ammonia water or 1% trifluoroacetic acid solution), preparation was carried out. Subsequently, the reaction system was purified by reverse-phase chromatography [mobile phase A1: 0.1% trifluoroacetic acid solution; mobile phase B: preparative acetonitrile; chromatography column inner diameter: 600 mm; chromatography column height: 23.0 cm; detection wavelength (monitoring wavelength): 210 nm (254 nm)] to obtain a solution containing mazdutide (purity ≥ 85.0%). Next, the solution was adjusted to a pH of 7.0–7.5 using 3% diluted ammonia water at 10–20 ℃, concentrated until no fractions remained at a reaction system temperature T ≤20 ℃ and reference P ≤-0.08 MPa, sampled for detection until the acetonitrile content reached 10%, and prepared by stirring for 1–2 hours to ensure uniform mixing. Subsequently, the reaction system was purified by reverse-phase chromatography [mobile phase A2: 50 mM ammonium bicarbonate aqueous solution; mobile phase B: preparative acetonitrile; chromatography column inner diameter: 600 mm; chromatography column height: 23.0 cm; detection wavelength (monitoring wavelength): 210 nm (254 nm)] to obtain a solution containing mazdutide (purity ≥ 97.0%).

[0463] Salt movement

[0464] The sample components purified by chromatography were diluted to twice the mass of purified water, and the reaction system was stirred for 1 to 2 hours. Subsequently, salt transfer preparation was performed using mobile phase A3 [preparative acetonitrile:water = 5:95 (v:v)], mobile phase A4 [preparative acetonitrile:32 g / L aqueous ammonium acetate = 5:95 (v:v)], mobile phase B (preparative acetonitrile), and a chromatography column (chromatography column inner diameter: 600 mm; chromatography column height: 23.0 cm) according to the following table. The detection wavelength (monitoring wavelength) was 210 nm (254 nm).

[0465]

[0466] Concentration and freeze-drying

[0467] Concentration: The components following purification by chromatography or salt migration in an enamel reactor were cooled to 10–20 °C, and the reaction system was adjusted to a pH of 7.0–7.5 using prepared 3% diluted ammonia water. The reaction system was concentrated until no fractions remained while controlling the internal temperature T to ≤20 °C and the reference pressure P to ≤-0.08 MPa, and sampling was performed for detection until the acetonitrile content reached ≤1.0%. The reaction system was purged with nitrogen below the liquid surface while controlling the temperature T to ≤20 °C, and sampling was performed every 4–8 hours until the residual acetonitrile reached ≤3000 ppm. To detect the pH value, the concentrated reaction system was sampled to adjust the pH of the solution to 7.0–7.5 (the pH of the solution can be adjusted with 3% diluted ammonia water).

[0468] Freeze-drying: The concentrated solution was pumped into freeze-drying trays at a rate of 1.8–2.4 kg / tray, and after filling was completed, primary freeze-drying was performed according to the following parameters.

[0469]

[0470] Concentration: The solid after freeze-drying was dissolved in 30 times its mass of purified water in an enamel reactor (the solution was adjusted to pH 7.0–7.5 with a prepared 3% diluted ammonia solution), and the reaction system was stirred while maintaining the pH at 7.0–7.5 until the solid was completely dissolved, and the acetonitrile content was detected until the acetonitrile content was ≤200 ppm (if the residual acetonitrile amount was >200 ppm, the reaction system could be purged with nitrogen below the liquid surface).

[0471] Freeze-drying: The concentrated solution was pumped into freeze-drying trays at a rate of 1.8–2.4 kg / tray, and after filling was completed, secondary freeze-drying was performed according to the following parameters.

[0472]

[0473] Dissolution: The solid after freeze-drying was dissolved in 30 times its mass in purified water in an enamel reactor (the solution was adjusted to pH 7.0–7.5 with a prepared 3% diluted ammonia solution), and the reaction system was stirred while maintaining the pH at 7.0–7.5 until the solid was completely dissolved, and the acetonitrile content was detected until the acetonitrile content was ≤200 ppm (if the residual acetonitrile amount was >200 ppm, the reaction system could be purged with nitrogen below the liquid surface).

[0474] Freeze-drying: The concentrated solution was pumped into freeze-drying trays at a rate of 1.8–2.4 kg / tray, and after filling was completed, tertiary freeze-drying was performed according to the following parameters.

[0475]

[0476] The product was recovered and verified, with a purity of 98.5% and a content of 88% to 95%.

[0477] Sequence list

[0478] Sequence No. 1

[0479]

[0480] Here, R1 and R3 are base stability side chain protectors, and

[0481] R2 is an ivDde side chain protector.

[0482] Sequence No. 2

[0483] H-Aib-QGTFTSDYSKYLDEKKAKEFV-EWLLEGGSSG-NH2

[0484] Here, the ε-amino group of the Lys side chain at position 20 is ([2-(2-aminoethoxy)-ethoxy]-acetyl)-2-(γ-Glu)-CO-(CH2) 18 It is chemically modified by conjugating to CO2H.

[0485] -10~0 ℃, 18~24 ℃, for 110~130 minutes

[0486] Sequence No. 3

[0487]

[0488] Sequence No. 4

[0489]

[0490] Sequence No. 5

[0491]

[0492] Sequence number 6

[0493]

[0494] Sequence No. 7

[0495]

[0496] Sequence No. 8

[0497]

Claims

Claim 1 A method for preparing a compound of the following chemical formula (sequence number 1), A method comprising the solid-phase synthesis of the above compound, wherein Fmoc-Gly-Gly-OH is used in the solid-phase synthesis, R1 and R3 are base stability side chain protecting groups, and R2 is an ivDde side chain protecting group. Claim 2 A method according to claim 1, wherein the solid phase synthesis is performed on a Fmoc amide resin solid support, and the Fmoc amide resin is a Rink amide resin. Claim 3 A method according to claim 1 or 2, wherein R1 is (a) Boc for Trp and Lys, (b) OtBu for Asp and Glu, (c) tBu for Ser, Thr and Tyr, (d) Trt for Gln and (e) Trt for His, and R3 is Boc, bonded to the amino group of His. Claim 4 In any one of claims 1 to 3, the solid-phase synthesis is performed on a Fmoc amide resin solid support, and the Fmoc deprotection step of the amide resin and the following Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH·H2O, Fmoc-Gly-Gly-OH, Fmoc-Glu(OtBu)-OH·H2O, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(OtBu)-OH·H2O, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH·H2O, Fmoc-Lys(ivDde)-OH, Fmoc-Ala-OH·H2O, A method comprising sequential coupling steps of Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Glu(OtBu)-OH·H2O, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Gln(Trt)-OH, Fmoc-Aib-OH and Boc-His(Trt)-OH. Claim 5 A method according to any one of claims 1 to 4, wherein the solid-phase synthesis comprises deprotection, activation, and coupling steps. Claim 6 In claim 5, the deprotection step is performed using a Pip / DMF / NMP solution or a DBU deprotection solution. Claim 7 A method according to claim 5 or 6, wherein the deprotection reaction during the solid-phase synthesis comprises the step of performing deprotection twice for 14 to 16 minutes each in the first 10 solid-phase synthesis cycles and the step of performing deprotection twice for 29 to 31 minutes each in the last 13 to 33 solid-phase synthesis cycles. Claim 8 A method according to claim 5 or 6, wherein the activation reaction system used in the activation step is DIC / HOBt / HOAt / Oxyma in DMF or DMF / NMP, or PyBOP / HOBt in DMF or DMF / NMP, or DEPBT in DMF or DMF / NMP. Claim 9 A method according to claim 5 or 6, wherein the coupling step is performed using 2 to 4 equivalents of Fmoc amino acids with respect to the resin in the first 10 solid-phase synthesis cycles, and in the remaining solid-phase synthesis cycles, the coupling step is performed using 4 to 6 equivalents of Fmoc amino acids with respect to the resin, or the coupling step is performed at His at position 1 and Lys at position 20 using 3 equivalents of Fmoc amino acids. Claim 10 A method according to claim 5 or 6, wherein the coupling step is performed at a temperature of 20 to 30 ℃ for a total reaction time of ≤12 hours. Claim 11 A method according to claim 5 or 6, wherein the solid phase synthesis optionally further comprises a terminal capping step. Claim 12 A method for preparing a compound of the following chemical formula (SEQ No. 2), wherein the Lys at position 20 has an ε-amino group of the Lys side chain at position 20 ([2-(2-aminoethoxy)-ethoxy]-acetyl)-2-(γ-Glu)-CO-(CH2) 18 It is chemically modified by conjugating to CO2H, and the method is (i) a solid-phase synthesis step of a compound of the following chemical formula (Sequence No. 1), wherein Step (ii) in which Fmoc-Gly-Gly-OH is used in the above solid-phase synthesis, where R1 and R3 are base stability side chain protecting groups and R2 is an ivDde side chain protecting group, the selective deprotection step of Lys at position 20, and the tBuO-C obtained through DIC / HOSu 20 A method comprising the steps of: directly coupling the -γGlu(tBu)-AEEA-AEEA-OSu active ester to the ε-amino group of the Lys side chain at position 20; (iii) cleaving the compound on a solid support and removing the base stability side chain protector; and (iv) purifying the compound. Claim 13 In claim 12, the solid phase synthesis is performed on a Fmoc amide resin solid support, and the Fmoc amide resin is a link amide resin, method. Claim 14 In claim 12 or 13, R1 is (a) Boc for Trp and Lys, (b) OtBu for Asp and Glu, (c) tBu for Ser, Thr and Tyr, (d) Trt for Gln and (e) Trt for His, and R3 is Boc, bonded to the amino group of His. Claim 15 In any one of claims 12 to 14, step (i) of the solid-phase synthesis is performed on a Fmoc amide resin solid support, and the Fmoc deprotection step of the amide resin and the following Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH·H2O, Fmoc-Gly-Gly-OH, Fmoc-Glu(OtBu)-OH·H2O, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(OtBu)-OH·H2O, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH·H2O, Fmoc-Lys(ivDde)-OH, Fmoc-Ala-OH·H2O, A method comprising sequential coupling steps of Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Glu(OtBu)-OH·H2O, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Gln(Trt)-OH, Fmoc-Aib-OH and Boc-His(Trt)-OH. Claim 16 A method according to any one of claims 12 to 15, wherein the solid-phase synthesis comprises deprotection, activation, and coupling steps. Claim 17 In paragraph 16, the above deprotection step is performed using a Pip / DMF / NMP solution or a DBU deprotection solution, a method. Claim 18 A method according to claim 16 or 17, wherein the deprotection reaction during the solid-phase synthesis comprises the step of performing deprotection twice for 14 to 16 minutes each in the first 10 solid-phase synthesis cycles and the step of performing deprotection twice for 29 to 31 minutes each in the last 13 to 33 solid-phase synthesis cycles. Claim 19 A method according to claim 16 or 17, wherein the activation reaction system used in the activation step is DIC / HOBt / HOAt / Oxyma in DMF or DMF / NMP, or PyBOP / HOBt in DMF or DMF / NMP, or DEPBT in DMF or DMF / NMP. Claim 20 A method according to claim 16 or 17, wherein the coupling step is performed using 2 to 4 equivalents of Fmoc amino acids for the resin in the first 10 solid-phase synthesis cycles, and in the remaining solid-phase synthesis cycles, the coupling step is performed using 4 to 6 equivalents of Fmoc amino acids for the resin, or the coupling step is performed at His at position 1 and Lys at position 20 using 3 equivalents of Fmoc amino acids. Claim 21 A method according to claim 16 or 17, wherein the coupling step is performed at a temperature of 20 to 30 ℃ for a total reaction time of ≤12 hours. Claim 22 A method according to claim 16 or 17, wherein the solid-phase synthesis optionally further comprises a terminal capping step. Claim 23 A method according to any one of claims 12 to 22, wherein Lys at position 20 is selectively deprotected by contacting the compound (Sequence No. 1) with a solution containing hydrazine hydrate. Claim 24 A method according to any one of claims 12 to 23, wherein in step (iii), the peptide is cleaved in a resin with an acidic mixture consisting of trifluoroacetic acid, triisopropylsilane, dithiothreitol, and water, and the reaction system after cleaving is directly crystallized in MTBE. Claim 25 A method according to any one of claims 12 to 24, wherein step (iv) comprises purifying the solution of the compound of step (iii) through chromatographic purification. Claim 26 In paragraph 25, the chromatographic purification is performed by HPLC or reverse-phase HPLC. Claim 27 The method according to claim 25, wherein the purification step further comprises the steps of (a) obtaining a product by elution using an ammonium acetate / acetonitrile (ACN) / water mobile phase, performing salt substitution, and removing trifluoroacetic acid (TFA), (b) performing concentration and removing the solvent, and (c) performing freeze-drying. Claim 28 A compound of the following formula I (SEQ ID NO. 3) or a pharmaceutically acceptable salt thereof, a compound of the following formula II (SEQ ID NO. 4) or a pharmaceutically acceptable salt thereof, or a compound of the following formula III (SEQ ID NO. 5) or a pharmaceutically acceptable salt thereof: Chemical formula I; Chemical formula II; Chemical formula III. Claim 29 In paragraph 28, the compound of Formula I or its pharmaceutically acceptable salt, the compound of Formula II or its pharmaceutically acceptable salt, and the compound of Formula III or its pharmaceutically acceptable salt are compounds or pharmaceutically acceptable salts selected from the following compounds or their pharmaceutically acceptable salts: Compound 2 (Sequence No. 6); Compound 3 (Sequence No. 7); Compound 4 (Sequence No. 8). Claim 30 As a compound of the following chemical formula (sequence number 1), The above compound is a compound prepared by a method according to any one of claims 1 to 11. Claim 31 As a compound of the following chemical formula (SEQ No. 2), H-Aib-QGTFTSDYSKYLDEKKAKEFV-EWLLEGGPSSG-NH2, the Lys at position 20 has the ε-amino group of the Lys side chain at position 20 ([2-(2-aminoethoxy)-ethoxy]-acetyl)-2-(γ-Glu)-CO-(CH2) 18 A compound that is chemically modified by conjugating to CO2H, said compound being prepared by a method according to any one of claims 12 to 27.

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