TW202404996A - Process for preparing a glp-1/glucagon dual agonist - Google Patents

Abstract

Disclosed herein are intermediate preparations for the manufacture of glucagon and GLP-1 dual agonist compounds or pharmaceutically acceptable salts thereof. Also disclosed herein are processes for the manufacture of glucagon and GLP-1 dual agonist compounds by coupling two to four intermediate preparations via hybrid solid liquid phase synthesis.

Description

Method for preparing GLP-1/glucagon dual agonist

The present invention provides a method for synthesizing glucagon (GCG) and GLP-1 dual agonist peptides or pharmaceutically acceptable salts thereof via mixed solid-liquid phase synthesis (mixed SPPS-LPPS, also referred to herein as HSLPS). .

Over the past few decades, the incidence of diabetes has continued to increase. Type 2 diabetes ("T2D") is the most common form of diabetes, accounting for approximately 90% of all diabetes cases. T2D is characterized by high blood sugar levels caused by insulin resistance. Uncontrolled diabetes leads to several conditions that affect patient morbidity and mortality. The main cause of death in patients with diabetes is cardiovascular complications. One of the major risk factors for type 2 diabetes is obesity. It has been documented that reducing body fat results in improvements in comorbidities associated with obesity, including hyperglycemia and cardiovascular events. Therefore, effective glucose control and weight loss therapies are needed for better disease management.

GCG helps maintain glucose levels in the blood by binding to GCG receptors on liver cells, causing the liver to release glucose stored in the form of glycogen through glycolysis. When these stores are depleted, GCG stimulates the liver to synthesize additional glucose through gluconeogenesis. This glucose is released into the bloodstream, preventing the development of hypoglycemia.

GLP-1 has different biological activities compared with GCG. The functions of GLP-1 include stimulating insulin synthesis and secretion, inhibiting GCG secretion, and inhibiting food intake. GLP-1 has been shown to reduce hyperglycemia in diabetes. Several GLP-1 agonists have been approved for the treatment of T2D in humans, including exenatide, liraglutide, lixisenatide, albiglutide ) and dulaglutide. Such GLP-1 agonists are effective in glycemic control and have beneficial effects on body weight without the risk of hypoglycemia. However, weight loss was modest due to dose-dependent gastrointestinal side effects.

GCG and GLP-1 dual agonist peptides suitable for the treatment of T2D and obesity are described and claimed in US Patent No. 9,938,335 B2. Methods for producing such GCG and GLP-1 dual agonist peptides are described therein.

The large-scale preparation of pharmaceutically superior GCG and GLP-1 dual agonist peptides presents many technical challenges that may affect overall yield and purity. Methods that use harsh reaction conditions that are incompatible with peptide synthesis also need to be avoided.

International Patent Application Publication No. WO2021/252829 describes a method for synthesizing GCG and GLP-1 dual agonist peptides using linear solid-phase peptide synthesis (SPPS). However, alternative methods for producing GCG and GLP-1 dual agonist peptides and intermediates thereof are needed to achieve pharmaceutically superior production with commercially desirable purity and yields. Likewise, methods and stable intermediates are needed to efficiently provide GCG and GLP-1 dual agonist peptides while reducing purification steps.

The present invention seeks to satisfy these needs by providing novel intermediates and methods suitable for the manufacture of GCG and GLP-1 dual agonist peptide (SEQ ID NO: 1) or pharmaceutically acceptable salts thereof. The method of the present invention provides intermediate products and process reactions that embody a combination of advanced technologies, including efficient pathways, while maintaining high quality and purity while reducing resource intensity and minimizing waste streams.

In one embodiment, a method is provided for preparing a compound of the formula: H ₂ NH-Aib-QGTFTSDYSKYLDEKKA- K -EFVEWLLEGPSSG-NH ₂ wherein the lysine acid at position 20 (Lys/K) is formed by ionization The ε-amino group and ([2-(2-aminoethoxy)-ethoxy]-acetyl group of the amino acid side chain) ₂ -(γ-Glu)-CO-(CH ₂ ) _18- CO Chemical modification is carried out by combining ₂ H (SEQ ID NO: 1).

Methods of the present invention include HSLPS methods, wherein such methods use two to four intermediate formulations to prepare the compound of SEQ ID NO: 1. The term "formulation" as used herein refers to compounds, such as peptide fragments or fatty acid moieties, used in the synthesis of the compound of SEQ ID NO: 1.

In one embodiment, the method may include at least the step of coupling three intermediate formulations, wherein such formulations have structures as set forth in SEQ ID NO: 2, 3 and 5 or pharmaceutically acceptable salts thereof.

Alternatively, the method may include at least the step of coupling three intermediate formulations, wherein such formulations have structures as set forth in SEQ ID NOs: 2, 10 and 12 or pharmaceutically acceptable salts thereof.

Alternatively, the method may include at least the step of coupling three intermediate formulations, wherein such formulations have the structures set forth in SEQ ID NOs: 17, 18 and 12 or pharmaceutically acceptable salts thereof.

Alternatively, the method may include at least the step of coupling three intermediate formulations, wherein such formulations have structures as set forth in SEQ ID NOs: 17, 21 and 5 or pharmaceutically acceptable salts thereof.

Alternatively, the method may include at least the step of coupling three intermediate formulations, wherein such intermediate formulations have structures as set forth in SEQ ID NOs: 36, 37 and 12 or pharmaceutically acceptable salts thereof.

Alternatively, the method may include at least the step of coupling two intermediate formulations, wherein such formulations have the structures set forth in SEQ ID NO: 24 and 25 or a pharmaceutically acceptable salt thereof.

In the above method, the fatty acid moiety and linker can be attached to one intermediate prior to coupling to the various intermediates (i.e., chelation would occur prior to complete synthesis).

Alternatively, the fatty acid moiety can be attached to the peptide after various intermediates have been coupled (i.e., chelation would occur after complete synthesis and selective deprotection of Lys 20).

For example, the method may include at least the steps of coupling three intermediate formulations, wherein such formulations have structures as set forth in SEQ ID NO: 2, 7 and 5 or pharmaceutically acceptable salts thereof, followed by coupling having the following structures The fatty acid part: (Preparation 30).

Alternatively, the method may include at least the step of coupling three intermediate formulations, wherein such formulations have the structures set forth in SEQ ID NOs: 2, 14 and 12 or pharmaceutically acceptable salts thereof, followed by coupling the structure having formulation 30 fatty acid fraction.

Alternatively, the method may include at least the step of coupling two intermediate formulations, wherein such formulations have structures as set forth in SEQ ID NOs: 27 and 25, or pharmaceutically acceptable salts thereof, followed by coupling an agent having the structure of Formulation 30 Fatty acid fraction.

Alternatively, the method may include at least the step of coupling a formulation having the structure as set forth in SEQ ID NO: 30 or a pharmaceutically acceptable salt thereof, followed by coupling a fatty acid moiety having the structure of Formulation 30.

The above method may also include the step of synthesizing an intermediate formulation prior to the coupling step.

Therefore, in the above method, the intermediate preparations can be chemically coupled to each other or enzymatically coupled to each other to obtain the compound of SEQ ID NO: 1.

In addition to the above methods, the embodiments herein also include the intermediate formulations themselves (eg, SEQ ID NOs: 2 to 39), as well as compositions including these intermediate formulations.

Advantages of the methods herein include several method improvements, such as shorter peptide fragments initially generated via solid phase peptide synthesis or SPPS allowing for overall improved purity and higher yields via HSLPS.

Advantages of the methods herein include: coupling efficiency in SPPS depends not only on the actual residues involved in the chemical transformation, but also on the structure attached to the resin (i.e., solubility/aggregation of certain sequences, especially GCG The problem is well known); in the case of shorter fragments, greater pathway flexibility can be used to couple complex amino acid residues, and the fragment structure can be redesigned to solve more difficult transformations.

Advantages of the methods herein include improved impurity control strategies during synthesis, which may result in an improved final impurity profile of the crude peptide and simplified/reduced chromatography burden resulting in cost savings.

Advantages of the methods herein include that synthesis of shorter fragments via SPPS allows for reduced wash cycles, allows for reduced volumes of reagents, and allows for the use of greener solvents, resulting in reduced process quality intensity (PMI).

Advantages of the methods herein include a significant reduction in the risk of failure typical in the linear construction of long molecules, especially those containing GCG-like sequences, in the case of shorter fragments.

The advantages of the method in this article include that the combination of liquid phase synthesis and solid phase synthesis is more suitable for new manufacturing platforms, such as continuous chemical methods and the introduction of other innovative technologies. Advantages of the methods herein include flexibility in the supply chain and logistics of the manufacturing process by using several independent segments. Advantages of the methods herein include that using parallel fabrication segments may provide reduced manufacturing cycle times by processing segments in parallel. Advantages of the methods herein include that current Good Manufacturing Practice (cGMP) liquid phase steps can be performed at standard manufacturing facilities without the need for specialized equipment.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the synthesis, the preferred methods and materials are described herein.

Furthermore, reference to "an element" by the indefinite article "a" or "an" does not exclude the possibility that more than one element is present, unless the context clearly requires the presence of one and only one element. Therefore, the indefinite article "a" or "an" usually means "at least one".

This application claims rights under 35 U.S.C. §119(e) in U.S. Provisional Application No. 63/327,120, filed on April 4, 2022; the disclosure content of this application is incorporated herein by reference.

Abbreviations and Definitions Some abbreviations are defined as follows: “AEEA” refers to 2-[2-(2-amino-ethoxy)-ethoxy]-acetyl, “Aib” refers to α-aminoisobutyl Acid, "Alloc" means allyloxycarbonyl, "Boc" means tertiary butoxycarbonyl, "Bu" means butyl, "t-Bu" means tertiary butyl, "CTC" means Chlorotrityl chloride, "DCM" refers to dichloromethane, "DIC" refers to diisopropylcarbodiimide, "DMF" refers to dimethylformamide, "DMSO" refers to dimethyl "Dnp" means 2,4-dinitrophenyl, "DTT" means dithiothreitol, "Fmoc" means fluorenylmethoxycarbonyl chloride, "hr" means hours, "HFIP" means hexafluoroisopropanol, "IPA" means isopropyl alcohol, "IPAc" means isopropyl acetate, "min" means minutes, "Me" means nails, "MTBE" means Methyl-tertiary butyl ether, "MTT" means 4-methyltrityl ether, "oxyma" means ethyl cyanohydroxyiminoacetate, "OtBu" means tertiary butyl ester, "PG ” refers to the protecting group, “Pip” refers to piperidine, “PyBOP” refers to (benzotriazol-1-yloxy)tripyrrolidinium phosphonium hexafluorophosphate, “SPPS” refers to solid phase peptide synthesis, “TFA” refers to trifluoroacetic acid, “TIPS” refers to triisopropylsilane and “Trt” refers to trityl.

As used herein, "about" means within a statistically significant range of one or more values such as specified concentration, length, molecular weight, pH, sequence identity, time range, temperature, or volume. Such values or ranges may be within an order of magnitude of a given value or range, typically within 20%, more typically within 10%, and even more typically within 5%. The allowed variations covered by "about" will depend on the particular system under consideration, and can be readily understood by those skilled in the art.

As used herein, the term "protected" means that a protecting group is attached at the specified position. The skilled artisan will recognize that a variety of protecting groups are well known and that alternative protecting groups may be suitable for a particular method.

The present invention provides and therefore encompasses novel intermediate compounds (referred to herein as "formulations" or "intermediates"), such as peptide fragments and fatty acid moieties, suitable for the synthesis of compounds of SEQ ID NO:1 and the synthesis of SEQ ID NO:1 compounds or pharmaceutically acceptable salts thereof. Intermediate formulations herein may be prepared by various techniques known in the art. By way of example, a method using standard solid-phase peptide synthesis for two or more intermediate formulations, which are subsequently coupled in liquid phase, is illustrated in the following example. The specific synthetic steps of each of the described pathways or schemes can be combined in different ways to prepare the compounds described herein. Reagents and starting materials are readily available to those skilled in the art.

Methods Standard Solid Phase Peptide Synthesis of Intermediate Formulation : Intermediate peptide fragments herein may be prepared via any number of standard peptide synthesis methods known in the art (especially SPPS). SPPS construction is achieved using standard Fmoc peptide chemistry techniques using automated peptide synthesizers such as the Gyros Protein Technologies Symphony X synthesizer for sequential coupling. SPPS methods are well known in the art and need not be described in detail herein. See generally , "Fmoc Solid Phase Peptide Synthesis: A Practical Approach" (eds. Chan & White, Oxford University Press 2000), and Merrifield (1963) J. Am. Chem. Soc. 85:2149-2154.

For deprotection, the resin was expanded with DMF and then deprotected using 20% Pip/DMF (3×30 min). The subsequent Fmoc deprotection group uses continuous 20% Pip/DMF treatment, and additional processing sequences are used for more difficult deprotection groups.

After removal of the protecting group, the resin was washed with DMF. Amino acid preactivation was performed using DIC/Oxyma DMF solution at room temperature for 30 min. Coupling of the activated amino acid to the resin-bound peptide occurs within a specified time for each individual amino acid. After coupling is complete, DMF washes are performed after each coupling to remove excess reagents.

To isolate the final product, the resin-bound product was washed with DCM to remove DMF. The resin was washed with IPA to displace DCM, followed by MTBE, and the product was then dried in vacuo at 40°C. The resin-bound product was stored at low temperature (-20°C).

Soft cleavage procedure for peptides that retain protecting groups : Treat the peptide on the resin intermediate with 30% HFIP in DCM. The spent resin was filtered off and washed with DCM. Pour the combined filtrates into 7 to 10 volumes of cold (0°C) MTBE. The suspension was aged for 30 min at 0 °C, the resulting pellet was centrifuged and the clear solution was decanted. The residue was suspended in the same volume of MTBE and centrifuged again and the resulting suspension decanted. After decanting the clear MTBE solution, the precipitated peptide was dried in vacuum at 40°C overnight.

An alternative soft cleavage method is as follows: soft cleavage of peptides from the resin using a 1 to 5% TFA/DCM mixture. The resin was swollen with DCM (10 vol, 1 x 20 min) and drained. 1 to 5% TFA/DCM (10 vol) was added to the pre-swollen resin and the suspension was stirred at room temperature for 10 minutes. The solution was filtered and the filtrate was treated with pyridine (equimolar to the added TFA). The resin was treated twice more with 1 to 5% TFA/DCM (10 vol), and the filtrates were combined and each time treated with pyridine (equimolar to TFA). The filtrate was concentrated under reduced pressure and the resulting residue was dissolved in DMF and precipitated from cold (0°C) water (10 volumes relative to DMF). The precipitate was filtered, washed with additional water (4 to 5 volumes relative to DMF), and dried in vacuum at 40°C overnight.

Hard cleavage procedure to remove protective groups : peptides are cleaved from the resin using an acidic mixture of TFA/H ₂ O/TIPS/DTT in the following ratio: (0.93v/0.04v/0.03v/0.03w). Swell the resin with DCM (4 to 5 vol, 3 x 30 min) and drain. The lysis mixture (4 to 5 volumes) was added to the pre-swollen resin and the suspension was stirred at room temperature for 2 hours. The solution was filtered, and the resin was then washed with a small amount of DCM and combined with the lysis solution. Pour the resulting solution into 7 to 10 volumes of cold (0°C) MTBE. The suspension was aged for 30 min at 0 °C, the resulting pellet was centrifuged and the clear solution was decanted. The residue was suspended in the same volume of MTBE and centrifuged again and the resulting suspension decanted. After decanting the clear MTBE solution, the precipitated peptide was dried in vacuum at 40°C overnight.

Mixed Solid-Liquid Phase Synthesis Intermediate peptide fragments prepared via SPPS as described above can be combined in the liquid phase to obtain the dual agonist of SEQ ID NO: 1. The methods of HSLPS are well known in the art and need not be described in detail herein. See generally , U.S. Patent Application Publication No. 2011/0046349; and Albericio et al. (1997) Methods Enzymol. 289:313-336, Bray et al. (2003) Nature Rev. Drug Discovery 2:587-593, Dalcol et al. (1995) J. Org. Chem. 7575-60:7581, Gauthier et al. (1991) Tettrahedron Lett. 32: 577-580, Schneider et al. (2005) J. Peptide Sci. 11:744-753, Smith, Organic Synthesis (Academic Press 4th edition 2016) and Zhang et al. (2008) Org. Process Res. Dev. 12:101-110.

Briefly, HSLPS involves the independent synthesis of intermediate peptide fragments in the solid phase and their coupling in the liquid phase.

As used herein, a method of preparing a compound of SEQ ID NO: 1 includes at least the step of coupling the following three intermediate fragments or formulations, wherein such formulations have the structures set forth in SEQ ID NO: 2, 3, and 5. In some cases, fragments can be coupled in the following order: SEQ ID NO:2 to SEQ ID NO:3 to SEQ ID NO:5 (i.e., from C-terminus to N-terminus). In other cases and with appropriate protecting group strategies, the fragments can be coupled in a different order.

A typical coupling scheme is to combine nearly equimolar amounts of the two fragments in a polar solvent such as DMSO. The acid-bearing fragments can be activated with stoichiometric amounts of PYBOP and Hunigs base or with a related activation system. After an appropriate reaction time, DEA was used to remove the protecting group with FMOC. Water is then added to induce precipitation of the resulting peptide coupled product, which is filtered, isolated and dried.

Another method of preparing the compounds of SEQ ID NO: 1 includes at least the step of coupling the following three intermediate formulations, wherein such formulations have the structures set forth in SEQ ID NO: 2, 10 and 12. In some cases, fragments are coupled in the following order: SEQ ID NO:2 to SEQ ID NO:10 to SEQ ID NO:12 (i.e., from C-terminus to N-terminus). In other cases and with appropriate protecting group strategies, the fragments can be coupled in a different order.

Another method of preparing the compounds of SEQ ID NO: 1 includes at least the step of coupling the following three intermediate formulations, wherein such formulations have the structures set forth in SEQ ID NO: 17, 18 and 12. In some cases, fragments are coupled in the following order: SEQ ID NO: 17 to SEQ ID NO: 18 to SEQ ID NO: 12 (i.e., from C-terminus to N-terminus). In other cases and with appropriate protecting group strategies, the fragments can be coupled in a different order.

Another method of preparing the compound of SEQ ID NO: 1 includes at least the step of coupling the following three intermediate formulations, wherein such formulations have the structures set forth in SEQ ID NO: 17, 21 and 5. In some cases, fragments are coupled in the following order: SEQ ID NO: 17 to SEQ ID NO: 21 to SEQ ID NO: 5 (i.e., from C-terminus to N-terminus). In other cases and with appropriate protecting group strategies, the fragments can be coupled in a different order.

Another method of preparing the compounds of SEQ ID NO: 1 includes at least the step of coupling the following three intermediate formulations, wherein such formulations have the structures set forth in SEQ ID NO: 36, 37 and 12. In some cases, the fragments are coupled in the following order: SEQ ID NO:36 to SEQ ID NO:37 to SEQ ID NO:12 (i.e., from C-terminus to N-terminus). In other cases and with appropriate protecting group strategies, the fragments can be coupled in a different order.

Another method of preparing the compounds of SEQ ID NO: 1 includes at least the step of coupling two intermediate formulations, wherein such formulations have the structures set forth in SEQ ID NO: 24 and 25.

Alternatively, other methods of making the compounds of SEQ ID NO: 1 use the same disconnection as described above, but instead couple all the amino acid segments of the backbone first, and then introduce the fatty acid side chain moieties as the final chemical Transformation followed by global deprotection. Here, for example, a corresponding PG can be constructed at Lys20, which can be selectively removed in the presence of other PGs such as Boc, Dnp, tBu and/or Trt.

In some cases, a method of preparing a compound of SEQ ID NO: 1 includes at least the step of coupling the following intermediate formulation to Formulation 30, wherein such formulation has a structure as set forth in SEQ ID NO: 2, 7, and 5. In some cases, the fragments are coupled in the following order: SEQ ID NO:2 to SEQ ID NO:7 to SEQ ID NO:5 (i.e., C-terminus to N-terminus) and then coupled to Formulation 30. In other cases and with appropriate protecting group strategies, the fragments can be coupled in a different order.

Another method of preparing the compounds of SEQ ID NO: 1 includes at least the step of coupling the following intermediate formulations to Formulation 30, wherein such formulations have the structures set forth in SEQ ID NO: 2, 14, and 12. In some cases, the fragments are coupled in the following order: SEQ ID NO:2 to SEQ ID NO:14 to SEQ ID NO:12 (i.e., C-terminus to N-terminus) and then coupled to Formulation 30. In other cases and with appropriate protecting group strategies, the fragments can be coupled in a different order.

Another method of preparing the compounds of SEQ ID NO: 1 includes at least the step of coupling the following intermediate formulations to Formulation 30, wherein such formulations have the structures set forth in SEQ ID NO: 27 and 25. In some cases, the fragments are coupled in the following order: SEQ ID NO:27 to SEQ ID NO:25, followed by coupling to Formulation 30. In other cases and with appropriate protecting group strategies, the fragments can be coupled in a different order.

In some cases, a method of preparing a compound of SEQ ID NO: 1 includes at least the steps of coupling an intermediate formulation having a structure as set forth in SEQ ID NO: 30 with intermediate formulation 30.

In order to efficiently synthesize the intermediate products of SEQ ID NO:3, 10, 18, 21, 24 and 37 (chelated peptide fragments), the following synthesis was performed using fatty acid side chains Formulation 30 and Fmoc- L -Lys-OH amino acid linked to fatty side chain: Preparation 31

For improved purity and efficiency of SPPS, the following tetramers and pentamers (SEQ ID NO: 31, 32, 33, 34 and 35) can be used to prepare intermediate formulations 4, 11, 24 and 28 (SEQ ID NO: 5, 12, 25 and 29), in which the following structures can be synthesized via SPPS or liquid phase synthesis using amino acid building blocks:

Thus, in one embodiment, SEQ ID NO:5 is prepared by coupling SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33. In another embodiment, SEQ ID NO:5 is prepared by coupling SEQ ID NO:31, SEQ ID NO:34, and SEQ ID NO:35. In another embodiment, SEQ ID NO:12 is prepared by coupling SEQ ID NO:31 and SEQ ID NO:34. In another embodiment, SEQ ID NO:25 is prepared by coupling SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33. In another embodiment, SEQ ID NO:25 is prepared by coupling SEQ ID NO:31, SEQ ID NO:34, and SEQ ID NO:35. In another embodiment, SEQ ID NO:29 is prepared by coupling SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33. In another embodiment, SEQ ID NO:29 is prepared by coupling SEQ ID NO:31, SEQ ID NO:34, and SEQ ID NO:35.

The skilled artisan will recognize that a variety of protecting groups are well known and alternative protecting groups may be used for amino acids. For example, in Boc-H(Dnp)-Aib-Q(Trt)-G) (SEQ ID NO:31), trityl or Boc may be present on the histidine instead of Dnp.

The compounds of SEQ ID NO: 1 herein may be used in a number of therapeutic applications, such as for the treatment of obesity, type 2 diabetes, non-alcoholic fatty liver disease (NAFLD) and/or non-alcoholic steatotic hepatitis (NASH) in individuals. ) method.

The compound of SEQ ID NO: 1 can be prepared as described in the combined synthetic schemes 1 to 10 shown below. Examples are provided for purposes of illustration and not limitation.

Example Example 1 (Process 1):

Example 2 (Process 2):

Example 3 (Process 3):

Example 4 (Process 4):

Example 5 (Process 5):

Example 6 (Process 6):

Example 7 (Process 7):

Example 8 (Process 8):

Example 9 (Process 9):

Example 30 (Process 10)

In Example 1 (Scheme 1), preparation 1 (SEQ ID NO:2), preparation 2 (SEQ ID NO:3) and preparation 4 (SEQ ID NO:5) were prepared by SPPS; preparation 3 (SEQ ID NO:5) was prepared by LPPS. ID NO:4), Formulation 5 (SEQ ID NO:6); and Compound 1 (SEQ ID NO:1) was prepared by deprotecting Formulation 5 (SEQ ID NO:6).

In Example 2 (Scheme 2), preparation 1 (SEQ ID NO:2), preparation 6 (SEQ ID NO:7) and preparation 4 (SEQ ID NO:5) were prepared by SPPS; preparation 7 (SEQ ID NO:5) was prepared by LPPS. ID NO: 8), formulation 8 (SEQ ID NO: 9); formulation 30 is coupled to the Lys side chain of formulation 8, followed by a removal step to obtain compound 1 (SEQ ID NO: 1).

In Example 3 (Scheme 3), preparation 1 (SEQ ID NO:2), preparation 9 (SEQ ID NO:10) and preparation 11 (SEQ ID NO:12) were prepared by SPPS; preparation 10 (SEQ ID NO:12) was prepared by LPPS. ID NO:11), Formulation 12 (SEQ ID NO:13); and Compound 1 (SEQ ID NO:1) was prepared by deprotecting Formulation 12 (SEQ ID NO:13).

In Example 4 (Scheme 4), preparation 1 (SEQ ID NO:2), preparation 13 (SEQ ID NO:14) and preparation 11 (SEQ ID NO:12) were prepared by SPPS; preparation 14 (SEQ ID NO:12) was prepared by LPPS. ID NO: 15), formulation 15 (SEQ ID NO: 16); formulation 30 is coupled to the Lys side chain of formulation 15, followed by a removal step to obtain compound 1 (SEQ ID NO: 1).

In Example 5 (Scheme 5), preparation 16 (SEQ ID NO:17), preparation 17 (SEQ ID NO:18) and preparation 11 (SEQ ID NO:12) were prepared by SPPS; preparation 18 (SEQ ID NO:12) was prepared by LPPS ID NO:19), Formulation 19 (SEQ ID NO:20); and Compound 1 (SEQ ID NO:1) was prepared by deprotecting Formulation 19 (SEQ ID NO:20).

In Example 6 (Scheme 6), preparation 16 (SEQ ID NO:17), preparation 20 (SEQ ID NO:21) and preparation 4 (SEQ ID NO:5) were prepared by SPPS; preparation 21 (SEQ ID NO:5) was prepared by LPPS ID NO:22), Formulation 22 (SEQ ID NO:23); and Compound 1 (SEQ ID NO:1) was prepared by deprotecting Formulation 22 (SEQ ID NO:23).

In Example 7 (Scheme 7), Formulation 23 (SEQ ID NO:24), Formulation 24 (SEQ ID NO:25) were prepared by SPPS; Formulation 25 (SEQ ID NO:26) was prepared by LPPS, and then removed protecting group to form compound 1 (SEQ ID NO:1).

In Example 8 (Scheme 8), preparation 24 (SEQ ID NO:25) and preparation 26 (SEQ ID NO:27) were prepared by SPPS; preparation 27 (SEQ ID NO:28) was prepared by LPPS; preparation 30 and preparation Coupling of the Lys side chain of 27 followed by removal of the protecting group gave compound 1 (SEQ ID NO: 1).

In Example 9 (Scheme 9), formulation 28 (SEQ ID NO:29) was prepared by SPPS; mild deprotection was performed to remove MTT to form formulation 29 (SEQ ID NO:30); formulation 30 and formulation 29 were The Lys side chain was coupled, followed by a deprotecting step to obtain compound 1 (SEQ ID NO: 1).

In Example 30 (Scheme 10), Formulation 37 (SEQ ID NO:36), Formulation 38 (SEQ ID NO:37) and Formulation 11 (SEQ ID NO:12) were prepared by SPPS; Formulation 39 (SEQ ID NO:12) was prepared by LPPS ID NO:38) and Formulation 40 (SEQ ID NO:39), and Compound 1 (SEQ ID NO:1) was prepared by deprotecting Formulation 40 (SEQ ID NO:39).

General procedure : Swelling of resin : Add resin (0.500 mmol) to the reactor and swell with DMF (3×10 mL×20 min). Washing after removing Fmoc : After removing the protecting group, wash the resin with DMF (5×10 mL×2 min). Washing after coupling : After coupling, wash the resin with DMF (5×10 mL×2 min). Washing and drying of the resin : After the final coupling or removal of the protecting group, the resin was washed with DMF (5×10 mL×2 min), followed by DCM (5×10 mL×2 min), and in _N atmosphere Drain and dry to constant weight.

Example 10 : Synthesis of Formulation 1 (SEQ ID NO: 2) by Solid Phase Peptide Synthesis Formulation 1 Formulation 1 (SEQ ID NO:2) or a pharmaceutically acceptable salt thereof was synthesized by standard SPPS using Sieber amide resin (loading ratio of 0.75 mmol/g) under the conditions set forth below. Table 1 cycle Fmoc-AA-OH Gly(30)-Gly(34) Reagents for removing Fmoc and conditions for removing Fmoc Coupling reagent Coincidence time Comment 1 Fmoc-Gly-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 13v DMF 4h Starting with Sieber amide resin; 0.75 mmol/g loading 2 Fmoc-L-Ser(tBu)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 13v DMF 4h 3 Fmoc-L-Ser(tBu)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 13v DMF 4h 4 Fmoc-L-Pro-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 13v DMF 4h 5 Fmoc-Gly-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 13v DMF 8h After coupling, use 20%PIP/DMF for 5+20+30 min at 25°C to remove Fmoc

Auto-resin cleavage : Add 5% TFA/DCM (10 vol) to the resin and stir the reactor for 30 minutes. The reactor was drained and the resin was washed with DCM (2x5v). The filtrate was added to pre-cooled MTBE:heptane (1:1, 10 volumes relative to (wrt) lysis solution) and then centrifuged (3000 rpm x 10 min). The supernatant was discarded and fresh cold MTBE:heptane (5 volumes) was added and the mixture was centrifuged (3000 rpm x 5 min). Discard the supernatant and repeat the process again with fresh MTBE:heptane. The supernatant was discarded and the resulting material was placed in a vacuum oven at 34°C for 14 hours to obtain Formulation 1. Mass experimental value: 515.8 [M+H].

Example 11 : Synthesis of Formulation 2 (SEQ ID NO:3) by Solid Phase Peptide Synthesis Formulation 2 Formulation 2 (SEQ ID NO:3) or a pharmaceutically acceptable salt thereof was synthesized by standard SPPS using Fmoc-Gly-CTC resin (loading ratio of 0.84 mmol/g) under the conditions set forth below. Table 2 cycle Fmoc-AA-OH Glu(16)-Gly(29) w/ Formulation 31 Reagents for removing Fmoc and conditions for removing Fmoc Coupling reagent Coincidence time Comment 1 Fmoc-Gly-CTC N/A 0.84 mmol/g load N/A Add 595 mg resin/reactor (0.500 mmol) 2 Fmoc-L-Glu(OtBu)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 1h 3 Fmoc-L-Leu-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 2h 4 Fmoc-L-Lys(Boc)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 10h 5 Fmoc-L-Leu-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 2h 6 Fmoc-L-Trp(Boc)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 2h 7 Fmoc-L-Glu(OtBu)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 4h 8 Fmoc-L-Val-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 4h 9 Fmoc-L-Phe-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC=3.0/3.0/3.3 14v DMF 4h 10 Fmoc-L-Glu(OtBu)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 6h 11 Preparation 31 20%PIP/DMF 5+20+30 min, 25℃ AA/PyBOP/DIEA 2.0/2.0/4.0 14v DMF 12h Prepare formulation 31, PyBOP, DIEA solution in DMF and pre-activate at RT for 30 min, and then add directly to RV 12 Fmoc-L-Ala-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 2h 13 Fmoc-L-Lys(Boc)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 4h 14 Fmoc-L-Lys(Boc)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 4h 15 Fmoc-L-Glu(OtBu)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 4h

Auto-resin cleavage: Use 20% HFIP/DCM solution to soft-cleave the peptide intermediate from the resin. The peptide intermediate on the resin was swollen with DCM (2 x 15 min x 10 vol). The resin was then treated with 20% HFIP/DCM (5×20 min×5 vol) and the filtrates were combined. The filtrate was concentrated to 1/3 volume at 30°C and the volume was replaced with chloroform (×3). The solution was then concentrated under reduced pressure to form a viscous residue, which was then added dropwise to n-heptane cooled to -15°C with vigorous stirring. The precipitate was filtered off and the residue was washed with n-heptane (×3). The resulting solid was then dried in vacuo for at least 18 hours. Quality experimental value: 3306.98

Example 12 : Synthesis of Formulation 4 (SEQ ID NO:5) by Solid Phase Peptide Synthesis Formulation 4 Formulation 4 (SEQ ID NO:5) or a pharmaceutically acceptable salt thereof was prepared by standard SPPS using Fmoc-L-Asp(OtBu)-CTC resin (loading ratio of 0.67 mmol/g) as described below. synthesized under the conditions. table 3 cycle Fmoc-AA-OH His(1)-Asp(15) Reagents for removing Fmoc and conditions for removing Fmoc Coupling reagent Coincidence time Comment 1 Fmoc-L-Asp(OtBu)-CTC N/A 0.67 mmol/g load N/A Add 746 mg resin/reactor (0.500 mmol) 2 Fmoc-L-Leu-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 12v DMF 4h 3 Fmoc-L-Tyr(tBu)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 12v DMF 4h 4 Fmoc-L-Lys(Boc)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 12v DMF 4h 5 Fmoc-L-Ser(tBu)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 12v DMF 4h 6 Fmoc-L-Tyr(tBu)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 12v DMF 4h 7 Fmoc-L-Asp(OtBu)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 12v DMF 4h 8 Fmoc-L-Ser(tBu)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 12v DMF 4h 9 Fmoc-L-Thr(tBu)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 12v DMF 4h 10 Fmoc-L-Phe-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 12v DMF 4h 11 Fmoc-L-Thr(tBu)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 12v DMF 4h 12 Fmoc-Gly-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 12v DMF 4h 13 Fmoc-L-Gln(Trt)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 12v DMF 10h 14 Fmoc-Aib-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 12v DMF 10h 15 Boc-L-His(Dnp)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 12v DMF 8h Prepare 4 mL of 0.375 M His solution using 1.50 mmol His and DMF and add directly to RV. Add 2 mL of 0.750 M Oxyma, followed by 2.5 mL of 0.660 M DIC, and continue mixing.

Auto-resin cleavage : Use 20% HFIP/DCM solution to soft-cleave the peptide intermediate from the resin. The peptide intermediate on the resin was swollen with DCM (2 x 15 min x 10 vol). The resin was then treated with 20% HFIP/DCM (5×20 min×5 vol) and the filtrates were combined. The filtrate was concentrated to 1/3 volume at 30°C and the volume was replaced with chloroform (×3). The solution was then concentrated under reduced pressure to form a viscous residue, which was then added dropwise to n-heptane cooled to -15°C with vigorous stirring. The precipitate was filtered off and the residue was washed with n-heptane (×3). The resulting solid was then dried in vacuo for at least 18 hours. Quality experimental value: 2802.51

Example 13 : Synthesis of Formulation 6 (SEQ ID NO:7) by Solid Phase Peptide Synthesis Formulation 6 Formulation 6 (SEQ ID NO:7) or a pharmaceutically acceptable salt thereof was synthesized by standard SPPS using Fmoc-Gly-CTC resin (loading ratio of 0.84 mmol/g) under the conditions set forth below. Table 4 cycle Fmoc-AA-OH Glu(16)-Gly(29) w/Lys(Alloc) Reagents for removing Fmoc and conditions for removing Fmoc Coupling reagent Coincidence time Comment 1 Fmoc-Gly-CTC N/A 0.84 mmol/g load N/A Add 595 mg resin/reactor (0.500 mmol) 2 Fmoc-L-Glu(OtBu)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 1h 3 Fmoc-L-Leu-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 2h 4 Fmoc-L-Lys(Boc)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 10h 5 Fmoc-L-Leu-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 2h 6 Fmoc-L-Trp(Boc)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 2h 7 Fmoc-L-Glu(OtBu)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 4h 8 Fmoc-L-Val-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 4h 9 Fmoc-L-Phe-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC=3.0/3.0/3.3 14v DMF 4h 10 Fmoc-L-Glu(OtBu)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 6h 11 Fmoc-L-Lys(Alloc)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC 3.0/3.0/3.3 14v DMF 12h 12 Fmoc-L-Ala-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 2h 13 Fmoc-L-Lys(Boc)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 4h 14 Fmoc-L-Lys(Boc)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 4h 15 Fmoc-L-Glu(OtBu)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 4h

Auto-resin cleavage: Use 20% HFIP/DCM solution to soft-cleave the peptide intermediate from the resin. The peptide intermediate on the resin was swollen with DCM (2 x 15 min x 10 vol). The resin was then treated with 20% HFIP/DCM (5×20 min×5 vol) and the filtrates were combined. The filtrate was concentrated to 1/3 volume at 30°C and the volume was replaced with chloroform (×3). The solution was then concentrated under reduced pressure to form a viscous residue, which was then added dropwise to n-heptane cooled to -15°C with vigorous stirring. The precipitate was filtered off and the residue was washed with n-heptane (×3). The resulting solid was then dried in vacuo for at least 18 hours. Quality experimental value: 2535.41

Example 14 : Synthesis of Formulation 11 (SEQ ID NO: 12) by Solid Phase Peptide Synthesis Formulation 11 Formulation 11 (SEQ ID NO: 12) or a pharmaceutically acceptable salt thereof was prepared by standard SPPS using Fmoc-L-Asp(OtBu)-CTC resin (loading ratio of 0.67 mmol/g) as described below. synthesized under the conditions. table 5 cycle Fmoc-AA-OH His(1)-Asp(9) Reagents for removing Fmoc and conditions for removing Fmoc Coupling reagent Coincidence time Comment 1 Fmoc-L-Asp(OtBu)-CTC N/A 0.67 mmol/g load N/A Add 746 mg resin/reactor (0.500 mmol) 2 Fmoc-L-Ser(tBu)-OH 20%PIP/DMF 5+20+30+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 12v DMF 6h 3 Fmoc-L-Thr(tBu)-OH 20%PIP/DMF 5+20+30+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 12v DMF 12h 4 Fmoc-L-Phe-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 12v DMF 4h 5 Fmoc-Gly-Thr(ψMe,MePro)-OH 20%PIP/DMF 5+20+30+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 12v DMF 7h 6 Fmoc-L-Gln(Trt)-OH 20%PIP/DMF 5+20+30+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 12v DMF 14h 7 Fmoc-Aib-OH 20%PIP/DMF 5+20+30+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 12v DMF 5h 8 Boc-L-His(Dnp)-OH 20%PIP/DMF 5+20+30+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 12v DMF 8h Prepare 4 mL of 0.375 M His solution using 1.50 mmol His and DMF and add directly to RV. Add 2 mL of 0.750 M Oxyma, followed by 2.5 mL of 0.660 M DIC, and continue mixing. *Boc-L-His(Boc)-OH can be used in cycle 8 to prepare fragments containing Boc-His(Boc).

Auto-resin cleavage : In a sintered reactor equipped with an overhead stirrer, add DCM (10v) to the resin and stir the reactor for 10 minutes to swell the resin. The reactor was drained and 1% TFA/DCM (10 vol) was added to the resin. The reactor was stirred for 10 minutes and the reactor was drained and the filtrate collected. Add fresh 1% TFA/DCM (10 vol) to the reactor and stir the reactor for 10 minutes. Pyridine (1:1 equivalent to the added TFA) was added to the filtrate. Drain the reactor and combine with the previous filtrate. Fresh 1% TFA/DCM (10 vol) was added to the reactor again and the reactor was stirred for 10 minutes. Additional pyridine (1:1 equivalent to the added TFA) was added to the filtrate. Drain the reactor and combine the filtrates. Pyridine (1:1 equivalent to the added TFA) was added to the filtrate. The reactor with spent resin was washed with DCM (2 x 10 volumes) and combined with the previous filtrate. The filtrate was concentrated under reduced pressure. The resulting residue was dissolved in minimal DMF and added dropwise to cold water (15 volumes relative to the DMF added to dissolve the residue) to precipitate the peptide. The suspension was filtered through a sintered funnel and the solids were rinsed with cold water (2 x 10 vol). The obtained solid was placed in a vacuum oven at 34°C for 14 hours to obtain fragments of formulation 11.

Contains Boc-His (Boc): M/Z experimental values = 1642.8911 and 1542.8396 [M-Boc]. Contains Boc-His(Dnp): M/Z experimental value=1708.8410

Example 15 : Synthesis of Formulation 16 (SEQ ID NO: 17) by Solid Phase Peptide Synthesis Formulation 16 Formulation 16 (SEQ ID NO: 17) or a pharmaceutically acceptable salt thereof was synthesized by standard SPPS using Sieber amide resin (loading ratio of 0.75 mmol/g) under the conditions set forth below. Table 6 cycle Fmoc-AA-OH Phe(22)-Gly(34) Reagents for removing Fmoc and conditions for removing Fmoc Coupling reagent Coincidence time Comment 1 Fmoc-Gly-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 1h Prepared from Sieber amide resin (0.75 mmol/g loading); add 667 mg resin (0.500 mmol) to the reactor 2 Fmoc-L-Ser(tBu)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 1h 3 Fmoc-L-Ser(tBu)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 1h 4 Fmoc-L-Pro-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 10h 5 Fmoc-Gly-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 1h 6 Fmoc-Gly-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 1h 7 Fmoc-L-Glu(OtBu)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 1h 8 Fmoc-L-Leu-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 2h 9 Fmoc-L-Leu-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC=3.0/3.0/3.3 14v DMF 10h 10 Fmoc-L-Trp(Boc)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 2h 11 Fmoc-L-Glu(OtBu)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC 3.0/3.0/3.3 14v DMF 2h 12 Fmoc-L-Val-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 4h 13 Fmoc-L-Phe-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 20h After coupling, use 20%PIP/DMF for 5+20+30 min at 25°C to remove Fmoc

Autoresin cleavage : Add resin (3 g) to a sinter reactor equipped with an overhead stirrer. The resin was suspended in DCM (28.5 mL) and TFA (1.5 mL) was added to the suspension. The resulting suspension was mixed for 30 minutes and the reactor was drained. The resin bed was washed with DCM (20 mL) and the filtrate was poured into pre-cooled MTBE:heptane (1:1, 300 mL). Centrifuge the suspension (3000 rpm x 10 min) and discard the supernatant. Add fresh pre-cooled MTBE:heptane (1:1, 300 mL) and centrifuge the suspension again (3000 rpm × 5 min). Discard the supernatant and repeat washing again with fresh MTBE:heptane (1:1, 300 mL). After centrifugation, the supernatant was discarded and the resulting solid was placed in a vacuum oven at 34°C for 14 hours to obtain formulation 16. Mass experimental value: 1699.9829.

Example 16 : Synthesis of Formulation 17 (SEQ ID NO:18) by Solid Phase Peptide Synthesis Formulation 17 (SEQ ID NO:18) or a pharmaceutically acceptable salt thereof was prepared by standard SPPS using as described below Conditional synthesis. Formulation 17 Formulation 17 (SEQ ID NO: 18) or a pharmaceutically acceptable salt thereof was prepared by standard SPPS using Fmoc-L-Glu(OtBu)-CTC resin (loading ratio of 0.655 mmol/g) as described below. synthesized under the conditions. Table 7 cycle Fmoc-AA-OH Tyr(10)-Glu(21) w/ Formulation 31 Reagents for removing Fmoc and conditions for removing Fmoc Coupling reagent Coincidence time Comment 1 Fmoc-L-Glu(OtBu)-CTC N/A 0.655 mmol/g load N/A To each reactor (0.500 mmol) add 763 mg 2 Preparation 31 20%PIP/DMF 5+20+30+30 min, 25℃ AA/PyBOP/DIEA= 3.0/3.0/6.0 12v DMF 14h To the vial was added 7.928 g of a 24.23% w/w solution of Formulation 31 in DMF, 781 mg of PyBOP was added, followed by 0.523 mL of DIEA and the solution was made up to 9 mL. Preactivate at room temperature for 30 minutes before adding to RV. 3 Fmoc-L-Ala-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 12v DMF 2h 4 Fmoc-L-Lys(Boc)-OH 20%PIP/DMF 5+20+30+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 12v DMF 14h 5 Fmoc-L-Lys(Boc)-OH 20%PIP/DMF 5+20+30+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 12v DMF 24h 6 Fmoc-L-Glu(OtBu)-OH 20%PIP/DMF 5+20+30+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 12v DMF 16h 7 Fmoc-L-Asp(OtBu)-OH 20%PIP/DMF 5+20+30+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 12v DMF 16h 8 Fmoc-L-Leu-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 12v DMF 4h 9 Fmoc-L-Tyr(tBu)-OH 20%PIP/DMF 5+20+30+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 12v DMF 14h 10 Fmoc-L-Lys(Boc)-OH 20%PIP/DMF 5+20+30+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 12v DMF 7h 11 Fmoc-L-Ser(tBu)-OH 20%PIP/DMF 5+20+30+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 12v DMF 12h 12 Fmoc-L-Tyr(tBu)-OH 20%PIP/DMF 5+20+30+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 12v DMF 5h

Auto-resin cleavage: In a sintered reactor equipped with an overhead stirrer, add DCM (10v) to the resin and stir the reactor for 10 minutes to swell the resin. The reactor was drained and 1% TFA/DCM (10 vol) was added to the resin. The reactor was stirred for 10 minutes and the reactor was drained and the filtrate collected. Add fresh 1% TFA/DCM (10 vol) to the reactor and stir the reactor for 10 minutes. Pyridine (1:1 equivalent to the added TFA) was added to the filtrate. Drain the reactor and combine with the previous filtrate. Fresh 1% TFA/DCM (10 vol) was added to the reactor again and the reactor was stirred for 10 minutes. Additional pyridine (1:1 equivalent to the added TFA) was added to the filtrate. Drain the reactor and combine the filtrates. Pyridine (1:1 equivalent to the added TFA) was added to the filtrate. The reactor with spent resin was washed with DCM (2 x 10 volumes) and combined with the previous filtrate. The filtrate was concentrated under reduced pressure. The resulting residue was dissolved in minimal DMF and added dropwise to cold water (15 volumes relative to the DMF added to dissolve the residue) to precipitate the peptide. The suspension was filtered through a sintered funnel and the solids were rinsed with cold water (2 x 10 vol). The obtained solid was placed in a vacuum oven at 34°C for 14 hours to obtain formulation 17. Mass experimental value: 1609.1 [M+H]

Example 17 : Synthesis of Formulation 32 (SEQ ID NO:31) by Solid Phase Peptide Synthesis Boc-H(Dnp)-Aib-Q(Trt)-G Formulation 32 (SEQ ID NO:31) Formulation 32 (SEQ ID NO:31) or a pharmaceutically acceptable salt thereof was prepared by standard SPPS using Fmoc-Gly-CTC resin (loading ratio of 1.04 mmol/g) as follows Synthesized under the conditions stated. Table 8 cycle Fmoc-AA-OH His(1)-Gly(4) Reagents for removing Fmoc and conditions for removing Fmoc Coupling reagent Coincidence time Comment 1 Fmoc-Gly-OH N/A CTC resin AA equivalent/DIEA equivalent=1.2/4.0 7 volume DCM 6h Load Fmoc-Gly-OH on CTC resin, and measure the resin loading (1.04 mmol/g) by qNMR 2 Fmoc-L-Gln(trt)-OH 20%PIP/DMF 4×30 min, 25℃ AA equivalent/Oxyma equivalent/DIC equivalent=3.0/3.0/3.3 13 volumes of DMF 12h 3 Fmoc-Aib-OH 20%PIP/DMF 4×30 min, 25℃ AA equivalent/Oxyma equivalent/DIC equivalent=3.0/3.0/3.3 13 volumes of DMF 12h 4 Boc-L-His(DNP)-OH 20%PIP/DMF 4×30 min, 25℃ AA equivalent/Oxyma equivalent/DIC equivalent=3.0/3.0/3.3 13v DMF 18h

Self-resin cleavage: Use 30% HFIP/DCM solution to softly cleave the peptide from the resin. The pre-expanded resin containing the Formulation 33 peptide was treated with 30% HFIP/DCM (10 vol) and stirred for 2 hours. The suspension was filtered and the resin cake was washed with DCM (4 to 5 volumes). The filtrate was concentrated under reduced pressure and then stripped with ACN three times to obtain Formulation 32 peptide as a dry foam.

Example 18 : Synthesis of Formulation 33 (SEQ ID NO:32) by Solid Phase Peptide Synthesis Fmoc-T(tBu)-FT-(tBu)-S(tBu) Formulation 33 (SEQ ID NO:32) Formulation 33 (SEQ ID NO:32) or a pharmaceutically acceptable salt thereof was prepared by standard SPPS using Fmoc-L-Ser(tBu)-CTC resin (0.71 mmol/g load ratio) is synthesized under the conditions described below. Table 9 cycle Fmoc-AA-OH Thr(5)-Ser(8) Reagents for removing Fmoc and conditions for removing Fmoc Coupling reagent Coincidence time Comment 1 Fmoc-L-Ser(tBu)-OH N/A CTC resin AA equivalent/DIEA equivalent = 0.8/3.0 7 volume DCM 18h Fmoc-L-Ser(tBu)-OH was loaded on CTC resin, and the resin loading (0.71 mmol/g) was measured by qNMR. 2 Fmoc-L-Thr(tBu)-OH 20%PIP/DMF 3×30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 13 volume DMF 4h 3 Fmoc-L-Phe-OH 20%PIP/DMF 3×30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 13 volume DMF 4h 4 Fmoc-L-Thr(tBu)-OH 20%PIP/DMF 3×30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 13 Volume DMF 4h

Self-resin cleavage: Use 30% HFIP/DCM solution to softly cleave the peptide from the resin. The pre-expanded resin containing the Formulation 33 peptide was treated with 30% HFIP/DCM (10 vol) and stirred for 2 hours. The suspension was filtered and the resin cake was washed with DCM (4 to 5 volumes). The filtrate was concentrated under reduced pressure and then stripped with ACN three times to obtain Formulation 33 peptide as a dry foam.

Example 19 : Synthesis of Formulation 34 (SEQ ID NO:33) by Solid Phase Peptide Synthesis Fmoc-D(tBu)-Y(tBu)-S(tBu)-K(Boc) Formulation 34 (SEQ ID NO:33) Formulation 34 (SEQ ID NO:33) or a pharmaceutically acceptable salt thereof was prepared by standard SPPS using Fmoc-L-Lys(Boc)-CTC resin (0.92 mmol/g load ratio) is synthesized under the conditions described below. Table 10 cycle Fmoc-AA-OH Asp(9)-Lys(12) Reagents for removing Fmoc and conditions for removing Fmoc Coupling reagent Coincidence time Comment 1 Fmoc-L-Lys(Boc)-OH N/A CTC resin AA equivalent/DIEA equivalent = 1.0/3.5 7 volume DCM 6h Fmoc-L-Lys(Boc)-OH was loaded on CTC resin, and the resin loading (0.92 mmol/g) was measured by qNMR. 2 Fmoc-L-Ser(tBu)-OH 20%PIP/DMF 3×30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 13 volume DMF 4h 3 Fmoc-L-Tyr(tBu)-OH 20%PIP/DMF 3×30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 13 volume DMF 4h 4 Fmoc-L-Asp(tBu)-OH 20%PIP/DMF 3×30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 13 volume DMF 4h

Auto-resin cleavage: Treat pre-swollen resin containing Formulation 34 peptide with 30% HFIP/DCM (10 vol) and stir for 2 hours. The suspension was filtered and the resin cake was washed with DCM (4 to 5 volumes). The filtrate was concentrated under reduced pressure and then stripped with ACN three times to obtain Formulation 34 peptide as a dry foam.

Example 20 : Synthesis of Formulation 35 (SEQ ID NO:34) by Solid Phase Peptide Synthesis Fmoc-T(tBu)-FT(tBu)-S(tBu)-D(tBu) Formulation 35 (SEQ ID NO:34) Formulation 35 (SEQ ID NO:34) or a pharmaceutically acceptable salt thereof was prepared by standard SPPS using Fmoc-L-Asp(tBu)-CTC resin (0.67 mmol/g load ratio) is synthesized under the conditions described below. Table 11 cycle Fmoc-AA-OH Thr(5)-Asp(9) Reagents for removing Fmoc and conditions for removing Fmoc Coupling reagent Coincidence time Comment 1 Fmoc-L-Asp(tBu)-OH N/A CTC resin AA equivalent/DIEA equivalent = 0.8/3.0 7 volume DCM 16h Fmoc-L-Asp(tBu)-OH was loaded on CTC resin, and the resin loading (0.67 mmol/g) was measured by qNMR. 2 Fmoc-L-Ser(tBu)-OH 20%PIP/DMF 3×30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 13 volume DMF 4h 3 Fmoc-L-Thr(tBu)-OH 20%PIP/DMF 3×30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 13 volume DMF 4h 4 Fmoc-L-Phe-OH 20%PIP/DMF 3×30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 13 volume DMF 4h 5 Fmoc-L-Thr(tBu)-OH 20%PIP/DMF 3×30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 13 volume DMF 4h

Auto-resin cleavage: Treat pre-expanded resin containing Formulation 35 peptide with 30% HFIP/DCM (10 vol) and stir for 2 hours. The suspension was filtered and the resin cake was washed with DCM (4 to 5 volumes). The filtrate was concentrated under reduced pressure and then stripped with ACN three times to obtain Formulation 35 peptide as a dry foam.

Example 21 : Synthesis of Preparation 31 Preparation 31 or ((52S)-52-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-25-(tertiary butoxycarbonyl)- 2,2-dimethyl-4,23,28,37,46-pentaoxy-3,32,35,41,44-pentaoxa-24,29,38,47-tetraazatripenta Dodecane-53-acid) was prepared as described in WO2020/159949.

Example 22 : Synthesis of Formulation 9 (SEQ ID NO: 10) by Solid Phase Peptide Synthesis Formulation 9 Formulation 9 (SEQ ID NO: 10) or a pharmaceutically acceptable salt thereof was synthesized by standard SPPS using H-Gly-CTC resin under the conditions set forth below. Table 12 cycle AA Conditions for removing protective groups Coupling conditions Coupling time (h) Comment 1 H-Gly-CTC - - - Resin with first AA preload 2 Fmoc-L-Glu(OtBu)-OH - AA/TBTU/DIPEA = 2 equivalents AA/1.8 equivalents TBTU/3.0 equivalents DIPEA 10 mL DMF per g starting resin 2 hours 3 Fmoc-L-Leu-OH 20%PIP/DMF time (10 + 20 min), ambient temperature AA/Oxyma/DIC= 2 equivalents AA/ 3.1 equivalents Oxyma/ 3.9 equivalents DIC 10 mL DMF per g starting resin 3 hours 4 Fmoc-L-Leu-OH 20%PIP/DMF time (10 + 20 min), ambient temperature AA/Oxyma/DIC= 2 equivalents AA/ 3.1 equivalents Oxyma/ 3.9 equivalents DIC 10 mL DMF per g starting resin 3 hours 5 Fmoc-L-Trp(Boc)-OH 20%PIP/DMF time (10 + 20 min), ambient temperature AA/Oxyma/DIC= 2 equivalents AA/ 3.1 equivalents Oxyma/ 3.9 equivalents DIC 10 mL DMF per g starting resin 3 hours 6 Fmoc-L-Glu(OtBu)-OH 20%PIP/DMF Time (10 + 20 min), ambient temp. AA/Oxyma/DIC= 2 equivalents AA/ 3.1 equivalents Oxyma/ 3.9 equivalents DIC 10 mL DMF per g starting resin 3 hours 7 Fmoc-L-Val-OH 20%PIP/DMF time (10 + 20 min), ambient temperature AA/Oxyma/DIC= 2 equivalents AA/ 3.1 equivalents Oxyma/ 3.9 equivalents DIC 10 mL DMF per g starting resin 3 hours 8 Fmoc-L-Phe-OH 20%PIP/DMF time (10 + 20 min), ambient temperature AA/Oxyma/DIC= 2 equivalents AA/ 3.1 equivalents Oxyma/ 3.9 equivalents DIC 10 mL DMF per g starting resin 3 hours 9 Fmoc-L-Glu(OtBu)-OH 20%PIP/DMF time (10 + 20 min), ambient temperature AA/Oxyma/DIC= 2 equivalents AA/ 3.1 equivalents Oxyma/ 3.9 equivalents DIC 10 mL DMF per g starting resin 3 hours 10 Preparation 31 20%PIP/DMF time (10 + 20 min), ambient temperature AA/Oxyma/DIC= 2 equivalents AA/ 3.1 equivalents Oxyma/ 3.9 equivalents DIC 10 mL DMF per g starting resin 3 hours 11 Fmoc-L-Ala-OH 20%PIP/DMF time (10 + 20 min), ambient temperature AA/Oxyma/DIC= 2 equivalents AA/ 3.1 equivalents Oxyma/ 3.9 equivalents DIC 10 mL DMF per g starting resin 3 hours 12 Fmoc-L-Lys(Boc)-OH 20%PIP/DMF time (10 + 20 min), ambient temperature AA/Oxyma/DIC= 2 equivalents AA/ 3.1 equivalents Oxyma/ 3.9 equivalents DIC 10 mL DMF per g starting resin 3 hours 13 Fmoc-L-Lys(Boc)-OH 20%PIP/DMF time (10 + 20 min), ambient temperature AA/Oxyma/DIC= 2 equivalents AA/ 3.1 equivalents Oxyma/ 3.9 equivalents DIC 10 mL DMF per g starting resin 3 hours 14 Fmoc-L-Glu(OtBu)-OH 20%PIP/DMF time (10 + 20 min), ambient temperature AA/Oxyma/DIC= 2 equivalents AA/ 3.1 equivalents Oxyma/ 3.9 equivalents DIC 10 mL DMF per g starting resin 3 hours 15 Fmoc-L-Asp(OtBu)-OH 20%PIP/DMF time (10 + 20 min), ambient temperature AA/Oxyma/DIC= 2 equivalents AA/ 3.1 equivalents Oxyma/ 3.9 equivalents DIC 10 mL DMF per g starting resin 4 hours 16 Fmoc-L-Leu-OH 20%PIP/DMF time (10 + 20 min), ambient temperature AA/Oxyma/DIC= 2 equivalents AA/ 3.1 equivalents Oxyma/ 3.9 equivalents DIC 10 mL DMF per g starting resin 3.3h 17 Fmoc-L-Tyr(tBu)-OH 20%PIP/DMF time (10 + 20 min), ambient temperature AA/Oxyma/DIC= 2 equivalents AA/ 3.1 equivalents Oxyma/ 3.9 equivalents DIC 10 mL DMF per g starting resin 3.25h 18 Fmoc-L-Lys(Boc)-OH 20%PIP/DMF time (10 + 20 min), ambient temperature AA/Oxyma/DIC= 2 equivalents AA/ 3.1 equivalents Oxyma/ 3.9 equivalents DIC 10 mL DMF per g starting resin 3.5 hours 19 Fmoc-L-Ser(tBu)-OH 20%PIP/DMF time (10 + 20 min), ambient temperature AA/Oxyma/DIC= 2 equivalents AA/ 3.1 equivalents Oxyma/ 3.9 equivalents DIC 10 mL DMF per g starting resin 3.25 hours 20 Fmoc-L-Tyr(tBu)-OH 20%PIP/DMF time (10 + 20 min), ambient temperature AA/Oxyma/DIC= 2 equivalents AA/ 3.1 equivalents Oxyma/ 3.9 equivalents DIC 10 mL DMF per g starting resin 3.25 hours

Auto-resin cleavage : Use 20% HFIP/DCM solution to soft-cleave the peptide intermediate from the resin. The peptide intermediate on the resin was swollen with DCM (2 x 15 min x 10 vol). The resin was then treated with 20% HFIP/DCM (5×20 min×5 vol) and the filtrates were combined. The filtrate was concentrated to 1/3 volume at 30°C and the volume was replaced with chloroform (×3). The solution was then concentrated under reduced pressure to form a viscous residue, which was then added dropwise to n-heptane cooled to -15°C with vigorous stirring. The precipitate was filtered off and the residue was washed with n-heptane (×3). The resulting solid was then dried in vacuo for at least 18 hours. Quality experimental value: 4400.64

Example 23 : Synthesis of Formulation 13 (SEQ ID NO: 14) by Solid Phase Peptide Synthesis Formulation 13 Formulation 13 (SEQ ID NO: 14) or a pharmaceutically acceptable salt thereof was synthesized by standard SPPS using H-Gly-CTC resin under the conditions set forth below. Table 13 cycle AA Conditions for removing protective groups Coupling conditions Coupling time (h) Comment 1 H-Gly-CTC - - - Resin with first AA preload 2 Fmoc-L-Glu(OtBu)-OH - AA/TBTU/DIPEA = 2 equivalents AA/1.8 equivalents TBTU/3.0 equivalents DIPEA 10 mL DMF per g starting resin 2 hours 3 Fmoc-L-Leu-OH 20%PIP/DMF time (10 + 20 min), ambient temperature AA/Oxyma/DIC= 2 equivalents AA/ 3.1 equivalents Oxyma/ 3.9 equivalents DIC 10 mL DMF per g starting resin 3 hours 4 Fmoc-L-Leu-OH 20%PIP/DMF time (10 + 20 min), ambient temperature AA/Oxyma/DIC= 2 equivalents AA/ 3.1 equivalents Oxyma/ 3.9 equivalents DIC 10 mL DMF per g starting resin 3 hours 5 Fmoc-L-Trp(Boc)-OH 20%PIP/DMF time (10 + 20 min), ambient temperature AA/Oxyma/DIC= 2 equivalents AA/ 3.1 equivalents Oxyma/ 3.9 equivalents DIC 10 mL DMF per g starting resin 3 hours 6 Fmoc-L-Glu(OtBu)-OH 20%PIP/DMF time (10 + 20 min), ambient temperature AA/Oxyma/DIC= 2 equivalents AA/ 3.1 equivalents Oxyma/ 3.9 equivalents DIC 10 mL DMF per g starting resin 3 hours 7 Fmoc-L-Val-OH 20%PIP/DMF time (10 + 20 min), ambient temperature AA/Oxyma/DIC= 2 equivalents AA/ 3.1 equivalents Oxyma/ 3.9 equivalents DIC 10 mL DMF per g starting resin 3 hours 8 Fmoc-L-Phe-OH 20%PIP/DMF time (10 + 20 min), ambient temperature AA/Oxyma/DIC= 2 equivalents AA/ 3.1 equivalents Oxyma/ 3.9 equivalents DIC 10 mL DMF per g starting resin 3 hours 9 Fmoc-L-Glu(OtBu)-OH 20%PIP/DMF time (10 + 20 min), ambient temperature AA/Oxyma/DIC= 2 equivalents AA/ 3.1 equivalents Oxyma/ 3.9 equivalents DIC 10 mL DMF per g starting resin 3 hours 10 Fmoc-L-Lys(Aloc)-OH 20%PIP/DMF time (10 + 20 min), ambient temperature AA/Oxyma/DIC= 2 equivalents AA/ 3.1 equivalents Oxyma/ 3.9 equivalents DIC 10 mL DMF per g starting resin 3 hours 11 Fmoc-L-Ala-OH 20%PIP/DMF time (10 + 20 min), ambient temperature AA/Oxyma/DIC= 2 equivalents AA/ 3.1 equivalents Oxyma/ 3.9 equivalents DIC 10 mL DMF per g starting resin 3 hours 12 Fmoc-L-Lys(Boc)-OH 20%PIP/DMF time (10 + 20 min), ambient temperature AA/Oxyma/DIC= 2 equivalents AA/ 3.1 equivalents Oxyma/ 3.9 equivalents DIC 10 mL DMF per g starting resin 3 hours 13 Fmoc-L-Lys(Boc)-OH 20%PIP/DMF time (10 + 20 min), ambient temperature AA/Oxyma/DIC= 2 equivalents AA/ 3.1 equivalents Oxyma/ 3.9 equivalents DIC 10 mL DMF per g starting resin 3 hours 14 Fmoc-L-Glu(OtBu)-OH 20%PIP/DMF time (10 + 20 min), ambient temperature AA/Oxyma/DIC= 2 equivalents AA/ 3.1 equivalents Oxyma/ 3.9 equivalents DIC 10 mL DMF per g starting resin 3 hours 15 Fmoc-L-Asp(OtBu)-OH 20%PIP/DMF time (10 + 20 min), ambient temperature AA/Oxyma/DIC= 2 equivalents AA/ 3.1 equivalents Oxyma/ 3.9 equivalents DIC 10 mL DMF per g starting resin 4.5 hours 16 Fmoc-L-Leu-OH 20%PIP/DMF time (10 + 20 min), ambient temperature AA/Oxyma/DIC= 2 equivalents AA/ 3.1 equivalents Oxyma/ 3.9 equivalents DIC 10 mL DMF per g starting resin 4.5 hours 17 Fmoc-L-Tyr(tBu)-OH 20%PIP/DMF time (10 + 20 min), ambient temperature AA/Oxyma/DIC= 2 equivalents AA/ 3.1 equivalents Oxyma/ 3.9 equivalents DIC 10 mL DMF per g starting resin 4.5 hours 18 Fmoc-L-Lys(Boc)-OH 20%PIP/DMF time (10 + 20 min), ambient temperature AA/Oxyma/DIC= 2 equivalents AA/ 3.1 equivalents Oxyma/ 3.9 equivalents DIC 10 mL DMF per g starting resin 4.5 hours 19 Fmoc-L-Ser(tBu)-OH 20%PIP/DMF time (10 + 20 min), ambient temperature AA/Oxyma/DIC= 2 equivalents AA/ 3.1 equivalents Oxyma/ 3.9 equivalents DIC 10 mL solvent per g starting resin: - Solvent = DMF (first coupling) - Solvent = NMP (second coupling) 2×4.5 h (repeated coupling) 20 Fmoc-L-Tyr(tBu)-OH 20%PIP/DMF time (10 + 20 min), ambient temperature AA/Oxyma/DIC= 2 equivalents AA/ 3.1 equivalents Oxyma/ 3.9 equivalents DIC 10 mL solvent per g starting resin: - Solvent = DMF (first coupling) - Solvent = NMP (second coupling) 2×6 h (repeated coupling) Additional Fmoc protection of all peptides was performed using 1.5 equiv Fmoc-Cl/2.0 equiv pyridine in DCM (7 mL DCM per g of starting resin) for a reaction time of 5 h.

Auto-resin cleavage : Use 20% HFIP/DCM solution to soft-cleave the peptide intermediate from the resin. The peptide intermediate on the resin was swollen with DCM (2 x 15 min x 10 vol). The resin was then treated with 20% HFIP/DCM (5×20 min×5 vol) and the filtrates were combined. The filtrate was concentrated to 1/3 volume at 30°C and the volume was replaced with chloroform (×3). The solution was then concentrated under reduced pressure to form a viscous residue, which was then added dropwise to n-heptane cooled to -15°C with vigorous stirring. The precipitate was filtered off and the residue was washed with n-heptane (×3). The resulting solid was then dried in vacuo for at least 18 hours. Quality experimental value: 3629.07

Example 24 : Synthesis of Formulation 20 (SEQ ID NO:21) by Solid Phase Peptide Synthesis Formulation 20 Formulation 20 (SEQ ID NO:21) or a pharmaceutically acceptable salt thereof was prepared by standard SPPS using Fmoc-L-Glu(OtBu)-CTC resin (loading ratio of 0.920 mmol/g) as described below synthesized under the conditions. Table 14 cycle Fmoc-AA-OH Glu(16)-Glu(21) w/ Formulation 31 Reagents for removing Fmoc and conditions for removing Fmoc Coupling reagent Coincidence time Comment 1 Fmoc-L-Glu(OtBu)-CTC N/A 0.920 mmol/g load N/A Add 54.4 g of resin to the reactor 2 Preparation 31 20%PIP/DMF (8v) 120 min, 25℃ AA/PyBOP/DIEA= 2.0/2.0/4.0 6v DMF 4h 3 Fmoc-L-Ala-OH 20%PIP/DMF (8v) 120 min, 25℃ AA/Oxyma/DIC= 2.0/2.0/2.2 6v DMF 4h 4 Fmoc-L-Lys(Boc)-OH 20%PIP/DMF (8v) 120 min, 25℃ AA/Oxyma/DIC= 2.0/2.0/2.2 6v DMF 4h 5 Fmoc-L-Lys(Boc)-OH 20%PIP/DMF (8v) 120 min, 25℃ AA/Oxyma/DIC= 2.0/2.0/2.2 6v DMF 4h 6 Fmoc-L-Glu(OtBu)-OH 20%PIP/DMF (8v) 120 min, 25℃ AA/Oxyma/DIC= 2.0/2.0/2.2 6v DMF 4h

Auto-resin cleavage : In a sintered reactor equipped with an overhead stirrer, add DCM (10v) to the resin and stir the reactor for 10 minutes to swell the resin. The reactor was drained and 1% TFA/DCM (10 vol) was added to the resin. The reactor was stirred for 10 minutes and the reactor was drained and the filtrate collected. Add fresh 1% TFA/DCM (10 vol) to the reactor and stir the reactor for 10 minutes. Pyridine (1:1 equivalent to the added TFA) was added to the filtrate. Drain the reactor and combine with the previous filtrate. Fresh 1% TFA/DCM (10 vol) was added to the reactor again and the reactor was stirred for 10 minutes. Additional pyridine (1:1 equivalent to the added TFA) was added to the filtrate. Drain the reactor and combine the filtrates. Pyridine (1:1 equivalent to the added TFA) was added to the filtrate. The reactor with spent resin was washed with DCM (2 x 10 volumes) and combined with the previous filtrate. The filtrate was concentrated under reduced pressure. The resulting residue was dissolved in minimal DMF and added dropwise to cold water (15 volumes relative to the DMF added to dissolve the residue) to precipitate the peptide. The suspension was filtered through a sintered funnel and the solids were rinsed with cold water (2 x 10 vol). The resulting solid was placed in a vacuum oven at 34°C for 14 hours to obtain fragments of formulation 20. Quality experimental value: 2121.2953

Example 25 : Synthesis of Formulation 36 (SEQ ID NO:35) by Solid Phase Peptide Synthesis Fmoc-Y(t-Bu)-S(t-Bu)-K(Boc)-Y(t-Bu) Formulation 36 (SEQ ID NO:35) Formulation 36 (SEQ ID NO:35) or a pharmaceutically acceptable salt thereof was prepared by standard SPPS using CTC resin (loading ratio of 0.671 mmol/g) under the conditions set forth below. Synthesis below. Table 15 cycle Fmoc-AA-OH Tyr(10)-Tyr(13) Reagents for removing Fmoc and conditions for removing Fmoc Coupling reagent Coincidence time Comment 1 Fmoc-L-Tyr(OtBu)-OH N/A CTC resin AA equivalent/DIEA equivalent = 1.2/7.8 7 volume DCM 14h Load Fmoc-L-Tyr(OtBu)-OH on CTC resin, and measure the resin loading by qNMR 2 Fmoc-L-Ser(tBu)-OH 20%PIP/DMF 3×30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 13 volume DMF 4h Starting with Fmoc-L-Tyr (OtBu) on CTC resin; loading of 0.671 mmol/g 3 Fmoc-L-Lys(Boc)-OH 20%PIP/DMF 3×30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 13 volume DMF 4h 4 Fmoc-L-Tyr(OtBu)-OH 20%PIP/DMF 3×30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 13 volume DMF 4h

Auto-resin cleavage : Treat pre-expanded resin containing Formulation 36 peptide with 30% HFIP/DCM (10 vol) and stir for 2 hours. The suspension was filtered and the resin cake was washed with DCM (4 to 5 volumes). The filtrate was concentrated under reduced pressure and then stripped with ACN three times to obtain Formulation 36 peptide as a dry foam.

Example 26 : Synthesis of Formulation 23 (SEQ ID NO:24) by Solid Phase Peptide Synthesis Formulation 23 Formulation 23 (SEQ ID NO:24) or a pharmaceutically acceptable salt thereof was synthesized by standard SPPS using Sieber amide resin (loading ratio of 0.75 mmol/g) under the conditions set forth below. Table 16 cycle Fmoc-AA-OH Lys(20)-Gly(34) w/ Formulation 31 Reagents for removing Fmoc and conditions for removing Fmoc Coupling reagent Coincidence time Comment 1 Fmoc-Gly-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 1h Prepared from Sieber amide resin (0.75 mmol/g loading); add 667 mg resin (0.500 mmol) to the reactor 2 Fmoc-L-Ser(tBu)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 1h 3 Fmoc-L-Ser(tBu)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 1h 4 Fmoc-L-Pro-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 10h 5 Fmoc-Gly-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 1h 6 Fmoc-Gly-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 1h 7 Fmoc-L-Glu(OtBu)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 1h 8 Fmoc-L-Leu-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 2h 9 Fmoc-L-Leu-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC=3.0/3.0/3.3 14v DMF 10h 10 Fmoc-L-Trp(Boc)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 2h 11 Fmoc-L-Glu(OtBu)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC 3.0/3.0/3.3 14v DMF 2h 12 Fmoc-L-Val-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 4h 13 Fmoc-L-Phe-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 20h 14 Fmoc-L-Glu(OtBu)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 6h 15 Preparation 31 20%PIP/DMF 5+20+30 min, 25℃ AA/PyBOP/DIEA=3.0/3.0/6.0 14v DMF 14h After coupling, use 20%PIP/DMF for 5+20+30 min at 25°C to remove Fmoc

Self-resin cleavage : Obtain a sample of the resin for hard cleavage to confirm the success of the build. Add 2.5 mL of lysis mix (2.32 mL TFA, 63 mg DTT, 0.063 mL TIPS, 0.063 mL H _x O) to the resin (200 mg) and mix the suspension for 2.5 hours. The resin was filtered off and rinsed with 1 mL of TFA, and the filtrate was poured into pre-cooled MTBE (17.5 mL). The suspension was aged at 0°C for 30 minutes and then centrifuged (3000 rpm x 10 min). The supernatant was discarded and the solid was washed with fresh MTBE (18 mL), centrifuged (3000 rpm x 5 min) and decanted. Repeat this step again with fresh MTBE and dry the resulting solid under vacuum for 14 hours. Mass experimental value: 1300.7 [M+2/2]

Example 27 : Synthesis of Formulation 24 (SEQ ID NO:25) by Solid Phase Peptide Synthesis Formulation 24 Formulation 24 (SEQ ID NO:25) or a pharmaceutically acceptable salt thereof was synthesized by standard SPPS using Fmoc-Ala-CTC resin, followed by soft cleavage from the resin.

Example 28 : Synthesis of Formulation 26 (SEQ ID NO:27) by Solid Phase Peptide Synthesis Formulation 26 Formulation 26 (SEQ ID NO: 27) or a pharmaceutically acceptable salt thereof was synthesized by standard SPPS using Sieber amide resin (loading ratio of 0.75 mmol/g) under the conditions set forth below. Table 17 cycle Fmoc-AA-OH Lys(20)-Gly(34) w/alloc Reagents for removing Fmoc and conditions for removing Fmoc Coupling reagent Coincidence time Comment 1 Fmoc-Gly-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 1h Prepared from Sieber amide resin (0.75 mmol/g loading); add 667 mg resin (0.500 mmol) to the reactor 2 Fmoc-L-Ser(tBu)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 1h 3 Fmoc-L-Ser(tBu)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 1h 4 Fmoc-L-Pro-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 10h 5 Fmoc-Gly-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 1h 6 Fmoc-Gly-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 1h 7 Fmoc-L-Glu(OtBu)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 1h 8 Fmoc-L-Leu-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 2h 9 Fmoc-L-Leu-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC=3.0/3.0/3.3 14v DMF 10h 10 Fmoc-L-Trp(Boc)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 2h 11 Fmoc-L-Glu(OtBu)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC 3.0/3.0/3.3 14v DMF 2h 12 Fmoc-L-Val-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 4h 13 Fmoc-L-Phe-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 20h 14 Fmoc-L-Glu(OtBu)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 6h 15 Fmoc-L-Lys(alloc)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 14h After coupling, use 20%PIP/DMF for 5+20+30 min at 25°C to remove Fmoc

Self-resin cleavage: Obtain a sample of the resin for hard cleavage to confirm the success of the build. Add 2.5 mL of lysis mix (2.32 mL TFA, 63 mg DTT, 0.063 mL TIPS, 0.063 mL H _x O) to the resin (200 mg) and mix the suspension for 2.5 hours. The resin was filtered off and rinsed with 1 mL of TFA, and the filtrate was poured into pre-cooled MTBE (17.5 mL). The suspension was aged at 0°C for 30 minutes and then centrifuged (3000 rpm x 10 min). The supernatant was discarded and the solid was washed with fresh MTBE (18 mL), centrifuged (3000 rpm x 5 min) and decanted. Repeat this step again with fresh MTBE and dry the resulting solid under vacuum for 14 hours. Quality experimental value: 1940.7

Example 29 : Synthesis of Formulation 29 (SEQ ID NO:30) by Solid Phase Peptide Synthesis Formulation 29 Formulation 29 (SEQ ID NO:30) or a pharmaceutically acceptable salt thereof was synthesized by standard SPPS using Sieber resin (loading ratio of 0.75 mmol/g) under the conditions set forth below. Table 18 cycle Fmoc-AA-OH His(1)-Gly(34) Reagents for removing Fmoc and conditions for removing Fmoc Coupling reagent Coincidence time Comment 1 Fmoc-Gly-OH 20%PIP/DMF 120 min, 25℃ AA/Oxyma/DIC= 2.0/2.0/2.2 5v DMF 2h Add Sieber resin to the reactor (loading = 0.75 mmol/g) 2 Fmoc-L-Ser(tBu)-OH 20%PIP/DMF 120 min, 25℃ AA/Oxyma/DIC= 2.0/2.0/2.2 5v DMF 2h 3 Fmoc-L-Ser(tBu)-OH 20%PIP/DMF 120 min, 25℃ AA/Oxyma/DIC= 2.0/2.0/2.2 5v DMF 2h 4 Fmoc-L-Pro-OH 20%PIP/DMF 120 min, 25℃ AA/Oxyma/DIC= 2.0/2.0/2.2 5v DMF 10h 5 Fmoc-Gly-OH 20%PIP/DMF 120 min, 25℃ AA/Oxyma/DIC= 2.0/2.0/2.2 5v DMF 2h 6 Fmoc-Gly-OH 20%PIP/DMF 120 min, 25℃ AA/Oxyma/DIC= 2.0/2.0/2.2 5v DMF 2h 7 Fmoc-L-Glu(OtBu)-OH 20%PIP/DMF 120 min, 25℃ AA/Oxyma/DIC= 2.0/2.0/2.2 5v DMF 4h 8 Fmoc-L-Leu-OH 20%PIP/DMF 120 min, 25℃ AA/Oxyma/DIC= 2.0/2.0/2.2 5v DMF 4h 9 Fmoc-L-Leu-OH 20%PIP/DMF 120 min, 25℃ AA/Oxyma/DIC= 2.0/2.0/2.2 5v DMF 10h 10 Fmoc-L-Trp(Boc)-OH 20%PIP/DMF 120 min, 25℃ AA/Oxyma/DIC= 2.0/2.0/2.2 5v DMF 4h 11 Fmoc-L-Glu(OtBu)-OH 20%PIP/DMF 120 min, 25℃ AA/Oxyma/DIC= 2.0/2.0/2.2 5v DMF 4h 12 Fmoc-L-Val-OH 20%PIP/DMF 360 min, 25℃ AA/Oxyma/DIC= 2.0/2.0/2.2 5v DMF 8h 13 Fmoc-L-Phe-OH 20%PIP/DMF 120 min, 25℃ AA/Oxyma/DIC= 2.0/2.0/2.2 5v DMF 6h 14 Fmoc-L-Glu(OtBu)-OH 20%PIP/DMF 120 min, 25℃ AA/Oxyma/DIC= 2.0/2.0/2.2 5v DMF 6h 15 Fmoc-Lys(Mtt)-OH 20%PIP/DMF 120 min, 25℃ AA/Oxyma/DIC= 2.0/2.0/2.2 5v DMF 14h 16 Fmoc-Ala-OH 20%PIP/DMF 120 min, 25℃ AA/Oxyma/DIC= 2.0/2.0/2.2 5v DMF 2h 17 Fmoc-Lys(Boc)-OH 20%PIP/DMF 120 min, 25℃ AA/Oxyma/DIC= 2.0/2.0/2.2 5v DMF 8h 18 Fmoc-Lys(Boc)-OH 20%PIP/DMF 120 min, 25℃ AA/Oxyma/DIC= 2.0/2.0/2.2 5v DMF 16h 19 Fmoc-Glu(OtBu)-OH 20%PIP/DMF 120 min, 25℃ AA/Oxyma/DIC= 2.0/2.0/2.2 5v DMF 16h 20 Fmoc-Asp(OtBu)-OH 20%PIP/DMF 120 min, 25℃ AA/Oxyma/DIC= 2.0/2.0/2.2 5v DMF 16h twenty one Fmoc-L-Leu-OH 20%PIP/DMF 120 min, 25℃ AA/Oxyma/DIC= 2.0/2.0/2.2 5v DMF 4h twenty two Fmoc-L-Tyr(tBu)-OH 20%PIP/DMF 120 min, 25℃ AA/Oxyma/DIC= 2.0/2.0/2.2 5v DMF 14h twenty three Fmoc-L-Lys(Boc)-OH 20%PIP/DMF 120 min, 25℃ AA/Oxyma/DIC= 2.0/2.0/2.2 5v DMF 8h twenty four Fmoc-L-Ser(tBu)-OH 20%PIP/DMF 120 min, 25℃ AA/Oxyma/DIC= 2.0/2.0/2.2 5v DMF 12h 25 Fmoc-L-Tyr(tBu)-OH 20%PIP/DMF 120 min, 25℃ AA/Oxyma/DIC= 2.0/2.0/2.2 5v DMF 6h 26 Fmoc-L-Asp(OtBu)-OH 20%PIP/DMF 120 min, 25℃ AA/Oxyma/DIC= 2.0/2.0/2.2 5v DMF 14h 27 Fmoc-L-Ser(tBu)-OH 20%PIP/DMF 120 min, 25℃ AA/Oxyma/DIC= 2.0/2.0/2.2 5v DMF 6h 28 Fmoc-L-Thr(tBu)-OH 20%PIP/DMF 120 min, 25℃ AA/Oxyma/DIC= 2.0/2.0/2.2 5v DMF 12h 29 Fmoc-L-Phe-OH 20%PIP/DMF 120 min, 25℃ AA/Oxyma/DIC= 2.0/2.0/2.2 5v DMF 4h 30 Fmoc-L-Thr(tBu)-OH 20%PIP/DMF 120 min, 25℃ AA/Oxyma/DIC= 2.0/2.0/2.2 5v DMF 10h 31 Fmoc-Gly-OH 20%PIP/DMF 120 min, 25℃ AA/Oxyma/DIC= 2.0/2.0/2.2 5v DMF 4h 32 Fmoc-L-Gln(Trt)-OH 20%PIP/DMF 120 min, 25℃ AA/Oxyma/DIC= 2.0/2.0/2.2 5v DMF 14h 33 Fmoc-Aib-OH 20%PIP/DMF 120 min, 25℃ AA/Oxyma/DIC= 2.0/2.0/2.2 5v DMF 6h 34 Boc-His(Boc)-OH 20%PIP/DMF 120 min, 25℃ AA/Oxyma/DIC= 2.0/2.0/2.2 5v DMF 8h Keep the histidine acid solution fresh before addition; do not pre-activate; add His solution directly to the reactor, then add Oxyma and DIC solutions, and start stirring for 8 hours

Soft cleavage of peptides from resin : Add resin (20 g) to a sintered reactor followed by DCM (10v). The resulting suspension was stirred with an overhead stirrer for 20 minutes to swell the resin. The reactor was drained and 20v of 5% TFA/DCM solution was added to the reactor and the resulting suspension was stirred for 30 minutes. The reactor was drained into a round bottom flask and the resin filter cake was washed with DCM (3x5v). Pyridine (equimolar to the added TFA) was added to the filtrate, and the filtrate was then concentrated under reduced pressure. The resulting orange-red residue was dissolved in 25 mL DMF and added dropwise to cold water (200 mL) to precipitate the peptide. Rinse the round bottom flask with 2 x 5 mL portions of DMF and add dropwise to the water/peptide suspension. The resulting suspension was placed in the refrigerator for 30 minutes and then filtered through a sintered funnel. The solid was washed with additional cold water and then placed in a vacuum oven at 35°C overnight, yielding 15.9 g of the desired peptide. Quality experimental value: 5444.1861 [M]; 5344.1325 [M-Boc]

Example 31 : Synthesis of Formulation 37 (SEQ ID NO:36) by Solid Phase Peptide Synthesis Formulation 37 Formulation 37 (SEQ ID NO:36) or a pharmaceutically acceptable salt thereof was synthesized by standard SPPS using Sieber amide resin (loading ratio of 0.75 mmol/g) under the conditions set forth below. Table 19 cycle Fmoc-AA-OH Glu(21)-Gly(34) Reagents for removing Fmoc and conditions for removing Fmoc Coupling reagent Coincidence time Comment 1 Fmoc-Gly-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 1h Prepared from Sieber amide resin (0.75 mmol/g loading); add 667 mg resin (0.500 mmol) to the reactor 2 Fmoc-L-Ser(tBu)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 1h 3 Fmoc-L-Ser(tBu)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 1h 4 Fmoc-L-Pro-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 10h 5 Fmoc-Gly-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 1h 6 Fmoc-Gly-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 1h 7 Fmoc-L-Glu(OtBu)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 1h 8 Fmoc-L-Leu-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 2h 9 Fmoc-L-Leu-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC=3.0/3.0/3.3 14v DMF 10h 10 Fmoc-L-Trp(Boc)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 2h 11 Fmoc-L-Glu(OtBu)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC 3.0/3.0/3.3 14v DMF 2h 12 Fmoc-L-Val-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 4h 13 Fmoc-L-Phe-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 20h 14 Fmoc-L-Glu(OtBu)-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 14v DMF 6h After coupling, use 20%PIP/DMF for 5+20+30 min at 25°C to remove Fmoc

Autoresin cleavage : Add resin (2 g) to a sinter reactor equipped with an overhead stirrer. The resin was suspended in DCM (19 mL) and TFA (1 mL) was added to the suspension. The resulting suspension was mixed for 30 minutes and the reactor was drained. The resin bed was washed with DCM (10 mL) and the filtrate was poured into pre-cooled MTBE:heptane (1:1, 200 mL). Centrifuge the suspension (3000 rpm x 10 min) and discard the supernatant. Add fresh pre-chilled MTBE:heptane (1:1, 200 mL) and centrifuge the suspension again (3000 rpm x 5 min). Discard the supernatant and repeat washing again with fresh MTBE:heptane (1:1, 200 mL). After centrifugation, the supernatant was discarded and the resulting solid was placed in a vacuum oven at 34°C for 14 hours to obtain Formulation 37. Quality experimental value: 1885.097

Example 32 : Synthesis of Formulation 38 (SEQ ID NO:37) by Solid Phase Peptide Synthesis Formulation 38 Formulation 38 (SEQ ID NO:37) or a pharmaceutically acceptable salt thereof was synthesized by standard SPPS using Formulation 31-CTC resin (loading ratio of 0.3574 mmol/g) under the conditions set forth below. Table 20 cycle Fmoc-AA-OH Tyr(10)-Lys(20) w/ Formulation 31 Reagents for removing Fmoc and conditions for removing Fmoc Coupling reagent Coincidence time Comment 1 Formulation 31-CTC N/A 0.3574 mmol/g load N/A Add 1.40 g of formulation 31-CTC (0.500 mmol) to the reactor 2 Fmoc-L-Ala-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 12v DMF 2h 3 Fmoc-L-Lys(Boc)-OH 20%PIP/DMF 5+20+30+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 12v DMF 14h 4 Fmoc-L-Lys(Boc)-OH 20%PIP/DMF 5+20+30+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 12v DMF 24h 5 Fmoc-L-Glu(OtBu)-OH 20%PIP/DMF 5+20+30+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 12v DMF 16h 6 Fmoc-L-Asp(OtBu)-OH 20%PIP/DMF 5+20+30+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 12v DMF 16h 7 Fmoc-L-Leu-OH 20%PIP/DMF 5+20+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 12v DMF 4h 8 Fmoc-L-Tyr(tBu)-OH 20%PIP/DMF 5+20+30+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 12v DMF 14h 9 Fmoc-L-Lys(Boc)-OH 20%PIP/DMF 5+20+30+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 12v DMF 7h 10 Fmoc-L-Ser(tBu)-OH 20%PIP/DMF 5+20+30+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 12v DMF 12h 11 Fmoc-L-Tyr(tBu)-OH 20%PIP/DMF 5+20+30+30 min, 25℃ AA/Oxyma/DIC= 3.0/3.0/3.3 12v DMF 5h

Auto-resin lysis : Add resin (1 g) to a 20 mL scintillation vial. The resin was suspended in DCM (7 mL) and HFIP (3 mL) was added to the suspension. Cap the vial and place it on a rotating wheel for 2 hours. The resin was filtered and the resin cake was washed with DCM (10 mL). The filtrate was concentrated under reduced pressure and the resulting residue was dissolved in MeCN and concentrated to dryness (×3) to obtain Formulation 38 as a sticky yellow solid. Quality experimental value: 3029.8636

Example 33 : Synthesis of Compound 1 Using Scheme 5 Liquid phase coupling of Formulation 16 and Formulation 17 to form Formulation 18: To a 2.5 wt% solution of Formulation 16 (97 mg) in DMF was added Formulation 17 (1.3 equiv) in DMF. 2.5wt% solution in DMF. The resulting solution was mixed and a 2.5 wt% solution of PyAOP (2.1 equiv) in MeCN was added to the mixture, followed by DIPEA (8.7 equiv). The resulting reaction mixture was stirred at room temperature for 2 hours before DEA (11.5 equiv) was added. The mixture was stirred at room temperature for 1 hour. Mass experimental value: 1559.8 [M+3/3]

Liquid phase coupling of Formulation 18 and Formulation 11 was performed to form Formulation 19: To a 5 wt% solution of Formulation 18 (40 mg) in DMF was added a 5 wt% solution of Formulation 11 (1.6 equiv) in DMF. The solutions were mixed and a 5 wt% solution of PyAOP (3.0 equiv) in MeCN was added to the solution, followed by the addition of a 5 wt% solution of DIPEA (8 equiv) in DMF. The resulting reaction mixture was stirred at room temperature for 2 hours and 40 minutes, and then 1.0 mL of 17% NaCl/0.5% _NaHCO solution was added and stirred for 5 minutes. Next, 1.0 mL of cold DI water was added, and the resulting mixture was stirred at 0°C for 1 hour. The white precipitate was then filtered through a sintered funnel and washed with DI water (2 mL). The obtained solid was dried in a vacuum oven at 30° C. overnight to obtain the desired substance in the form of an off-white solid.

Entire deprotection of Formulation 19 was performed to form Compound 1 (SEQ ID NO: 1): TFA (0.424 mL), TIPS (11.5 μL), water (11.5 μL) and DTT (11.5 mg) were added to the solid obtained above. The resulting mixture was mixed on a rotating wheel for 2 hours and then poured into 11 mL of cold MTBE. The resulting pellet was centrifuged at 3000 rpm for 3 minutes and washed twice with MTBE and centrifuged each time. The solid was dried at room temperature. Mass experimental value: 1521.9 [M+3/3]

Example 34 : Synthesis of Compound 1 Using Scheme 6 Liquid phase coupling of Formulation 16 and Formulation 20 to form Formulation 21: To a 250 mL round bottom flask equipped with a magnetic stir bar was added Formulation 16 (1.26 g, 1.02 equiv) . Add 5 mL portions of DMF/THF (85/15, 20 mL) to the flask. The material is kept stirred for about 5 minutes to completely dissolve the material. Simultaneously, Formulation 20 (2.10 g, 1.00 equiv) and PyAOP (450 mg, 1.56 equiv) were added to a 20 mL scintillation vial, and the material was dissolved in DMF/THF (85/15, 10 mL) and then added to In a round bottom flask. Rinse vial with 2 x 5 mL portions of DMF/THF (85/15). DIPEA (0.67 mL, 7.00 equiv) was added to the reaction mixture and the resulting reaction mixture was stirred at room temperature. After 18 hours, DEA (0.57 mL, 10 equiv) was added and the resulting solution was stirred for 1 hour. After 1 hour, the reaction mixture was added dropwise to a pre-cooled 17% NaCl/0.5% NaHCO ₃ aqueous solution (400 mL) to precipitate the peptide. The suspension was filtered and the solids washed with 2 x 50 mL cold DI water. Dry the solid in a vacuum oven at 34°C overnight. Quality experimental value: 3581.2002

Liquid phase coupling of Formulation 21 and Formulation 4 to form Formulation 22: To a solution of Formulation 21 (522 mg, 1.05 equiv) in DMF/THF (85/15, 8 mL) was added Formulation 4 (w/His(Dnp )) (385 mg, 1.02 equiv) and PyAOP (66 mg, 1.53 equiv) in DMF/THF (85/15, 3 mL). The solution was allowed to mix for 5 minutes to dissolve the material, and then DIPEA (0.044 mL, 3.00 equiv) was added to the reaction mixture. The resulting reaction mixture was allowed to stir at room temperature for 4 hours, and then added dropwise to 110 mL of pre-cooled 17% NaCl/0.5% NaHCO ₃ aqueous solution to precipitate the peptide. The suspension was then filtered through a sintered funnel and the solids washed with 2 x 25 mL DI water. The solid was then dried in a vacuum oven at 34°C for 14 hours. Quality experimental value: 6365.6991

Entire deprotection of Formulation 22 was performed to form Compound 1 (SEQ ID NO: 1): TFA (0.424 mL), TIPS (11.5 μL), water (11.5 μL) and DTT (11.5 mg) were added to the solid obtained above. The resulting mixture was mixed on a rotating wheel for 2 hours and then poured into 11 mL of cold MTBE. The resulting pellet was centrifuged at 3000 rpm for 3 minutes and washed twice with MTBE and centrifuged each time. The solid was dried at room temperature. Mass experimental value: 1521.9 [M+3/3]

Example 35 : Synthesis of Compound 1 using Scheme 9 Liquid phase coupling of Formulation 29 with Formulation 30 to form Compound 1 (SEQ ID NO: 1): Coupling of Formulation 30 with Formulation 29: Reaction equipped with a magnetic stir bar Add Formulation 29 (2 g) and DMF (15 mL) to the container. The resulting mixture was mixed until the peptide was completely dissolved (approximately 10 min). A solution of Formulation 30 (2.0 equiv) and PyBOP (2.0 equiv) in DMF (5 mL) was prepared and then added to the peptide solution. DIPEA (4.0 equiv) was added to the reaction mixture and the resulting reaction mixture was stirred for 18 hours. The reaction mixture was then added dropwise to 200 mL of pre-cooled 17% NaCl/0.5% NaHCO ₃ aqueous solution to precipitate the peptide. The resulting suspension was aged in the refrigerator for 30 minutes and then filtered through a sintered funnel. The solid was washed with additional cold water and then dried in a vacuum oven overnight to give the desired peptide (2.16 g). Quality experimental value: 6299.7459 [M]; 6199.6908 [M-Boc]

Global deprotection: Add 10v of lysis mixture (92.5% TFA:2.5% TIPS:2.5% DTT:2.5% H ₂ O) to the protected peptide (1 g). The resulting mixture was allowed to mix for 2 hours and then added dropwise to pre-cooled MTBE (7v) to precipitate the peptide. The suspension was centrifuged (3000 rpm x 10 min) and the supernatant discarded. The solid was then suspended in fresh MTBE (7v) and centrifuged again (3000 rpm x 5 min). Discard the supernatant and repeat the process again with fresh MTBE (7v). After centrifugation, the supernatant was discarded and the resulting solid was dried in a vacuum oven overnight to obtain crude peptide. Mass experimental value: 4560.2610.

Example 36 : Synthesis of Compound 1 Using Scheme 10 Liquid phase coupling of Formulation 37 and 38 to form Formulation 39: To Formulation 37 (54 mg, 1.18 equiv) in DMF/THF (85/15, 0.5 mL) A solution of Formulation 38 (73 mg, 1.00 equiv) and PyAOP (26 mg, 2.10 equiv) in DMF/THF (85/15, 3 mL) was added to the solution. The mixture was allowed to stir for approximately 5 minutes to allow the solids to dissolve, and then DIPEA (0.03 mL, 7.89 equiv) was added and the resulting reaction mixture was stirred. After 4 hours, DEA (0.03 mL, 11.3 equiv) was added and the resulting mixture was stirred at room temperature for one hour. The reaction mixture was then added dropwise to a pre-cooled 17% NaCl/0.5% NaHCO ₃ aqueous solution (35 mL) to precipitate the peptide. The suspension was filtered through a sintered funnel and the solids were washed with 2 x 10 mL DI water. The solid was dried in a vacuum oven at 34°C for 14 hours to obtain formulation 39 in 49% crude yield. Quality experimental value: 4674.8656

Liquid phase coupling of Formulation 39 and Formulation 11 to form Formulation 40: Formulations 39 and 11 were coupled in the liquid phase using the coupling conditions described in Scheme 5, second coupling step (Example 33).

Entire deprotection of Formulation 40 was performed to form Compound 1 (SEQ ID NO: 1): Overall deprotection of formulation 40 was performed using conditions described in Example 33.

Example 37 : Synthesis of Formulation 11 using Tetramer Formulation 32 and Formulation 33 Fmoc-Asp(OtBu)-CTC (0.500 mmol, 0.67 mmol/g) resin was added to the solid phase reactor and followed by DMF (3 ×10 mL × 20 min) for expansion, and then use 20% piperidine/DMF (3 × 10 mL × 30 min) to remove the protecting group. Add 4 mL of Formulation 33 (Fmoc-T(tBu)-FT(tBu)-S(tBu)-OH) 0.375 M in DMF to the preactivation vessel, followed by 2 mL of Oxyma 0.750 M in DMF M solution and 2.5 mL of a 0.660 M solution of DIC in DMF. The resulting solution was mixed for 30 minutes while bubbling _N2 and then transferred to a reactor containing the resin and coupled for 12 hours. The reactor was drained and the resin was then washed with DMF (5×10 mL×2 min). Fmoc was removed with 20% piperidine/DMF (3×10 mL×30 min), and the resin was then washed with DMF (5×10 mL×2 min). Add 4 mL of Formulation 32 (Boc-His(dnp)-Aib-Q(Trt)-G-OH) 0.375 M in DMF to the preactivation vessel, followed by 2 mL of Oxyma 0.750 M in DMF solution and 2.5 mL of a 0.660 M solution of DIC in DMF. The resulting solution was mixed for 30 minutes while bubbling _N2 and then transferred to a reactor containing the resin and coupled for 12 hours. The reactor was then drained, and the resin was washed with DMF (5×10 mL×2 min), followed by DCM (5×10 mL×2 min), and then drained to dryness under _N purge for 8 h. A sample of the resin is then hard-cracked (see Hard-Cracking Procedure) to confirm the success of the build. Quality experimental value: 1143.3.

Sequences The following sequences are mentioned in the present invention and are provided below for reference. H-Aib-QGTFTSDYSKYLDEKKAK((2-[2-(2-aminoethoxy)-ethoxy]-acetyl) ₂ -(γ-Glu)-CO-(CH ₂ ) ₁₈ -CO ₂ H )EFVEWLLEGPSSG-NH ₂ SEQ ID NO:31 Boc-H(Dnp)-Aib-Q(Trt)-G SEQ ID NO:32 Fmoc-T(tBu)-FT-(tBu)-S(tBu) SEQ ID NO:33 Fmoc-D (tBu)-Y(tBu)-S(tBu)-K(Boc) SEQ ID NO:34 Fmoc-T(tBu)-FT(tBu)-S(tBu)-D(tBu) SEQ ID NO:35 Fmoc -Y(t-Bu)-S(t-Bu)-K(Boc)-Y(t-Bu)

TW202404996A_112112452_SEQL.xml

Claims (27)

A method for preparing the compound of SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof, the method comprising coupling an intermediate preparation selected from the group consisting of: a. SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:12; b. SEQ ID NO:17, SEQ ID NO:21 and SEQ ID NO:5; c. SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:12; d. SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:5; e. SEQ ID NO:2, SEQ ID NO:10 and SEQ ID NO:12; f. SEQ ID NO:24 and SEQ ID NO:25; g. SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:5 and preparation 30; h. SEQ ID NO:2, SEQ ID NO:14, SEQ ID NO:12 and preparation 30; i. SEQ ID NO:27, SEQ ID NO:25 and Formulation 30; and j. SEQ ID NO:30 and Formulation 30. The method of claim 1, wherein the method comprises coupling SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:12. The method of claim 1, wherein the method includes coupling SEQ ID NO:17, SEQ ID NO:21 and SEQ ID NO:5. The method of claim 1, wherein the method comprises coupling SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:12. The method of claim 1, wherein the method includes coupling SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:5. The method of claim 1, wherein the method includes coupling SEQ ID NO:2, SEQ ID NO:10 and SEQ ID NO:12. The method of claim 1, wherein the method includes coupling SEQ ID NO: 24 and SEQ ID NO: 25. The method of claim 1, wherein the method comprises coupling SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:5 and formulation 30. The method of claim 1, wherein the method comprises coupling SEQ ID NO:2, SEQ ID NO:14, SEQ ID NO:12 and formulation 30. The method of claim 1, wherein the method comprises coupling SEQ ID NO:27, SEQ ID NO:25 and formulation 30. The method of claim 1, wherein the method comprises coupling SEQ ID NO: 30 and formulation 30. For example, the method of request item 2 includes: Each of SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:12 was synthesized by solid phase peptide synthesis and coupled in liquid phase. For example, the method of request item 3 includes: Each of SEQ ID NO:17, SEQ ID NO:21 and SEQ ID NO:5 was synthesized by solid phase peptide synthesis and coupled in liquid phase. For example, the method of request item 4 includes: Each of SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:12 was synthesized by solid phase peptide synthesis and coupled in liquid phase. For example, the method of request item 5 includes: Each of SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:5 was synthesized by solid phase peptide synthesis and coupled in liquid phase. For example, the method of request item 6 includes: Each of SEQ ID NO:2, SEQ ID NO:10 and SEQ ID NO:12 was synthesized by solid phase peptide synthesis and coupled in liquid phase. For example, the method of request item 7 includes: Each of SEQ ID NO:24 and SEQ ID NO:25 was synthesized by solid phase peptide synthesis and coupled in liquid phase. For example, the method of request item 8 includes: Each of SEQ ID NO:2, SEQ ID NO:7 and SEQ ID NO:5 was synthesized by solid phase peptide synthesis and coupled in the liquid phase, followed by coupling with Formulation 30 in the liquid phase. For example, the method of request item 9 includes: Each of SEQ ID NO:2, SEQ ID NO:14 and SEQ ID NO:12 was synthesized by solid phase peptide synthesis and coupled in the liquid phase, followed by coupling with Formulation 30 in the liquid phase. For example, the method of request item 10 includes: Each of SEQ ID NO:27 and SEQ ID NO:25 was synthesized by solid phase peptide synthesis and coupled in the liquid phase, followed by coupling with Formulation 30 in the liquid phase. Such as the method of request item 11, which method includes: SEQ ID NO:30 was synthesized by solid phase peptide synthesis and coupled to Formulation 30 in liquid phase. An intermediate preparation, which is selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO :30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36 and SEQ ID NO:37, or their pharmaceutical Take it with a pinch of salt. An intermediate preparation, which is selected from the group consisting of: SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:21 and SEQ ID NO:22, or pharmaceutically acceptable salts thereof. The method of claim 1, comprising preparing SEQ ID NO: 5 by coupling: a. SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:33; or b. SEQ ID NO:31, SEQ ID NO:34 and SEQ ID NO:35. The method of claim 1, comprising preparing SEQ ID NO:12 by coupling SEQ ID NO:31 and SEQ ID NO:34. The method of claim 1, comprising preparing SEQ ID NO: 25 by coupling: a. SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:33; or b. SEQ ID NO:31, SEQ ID NO:34 and SEQ ID NO:35. The method of claim 1, comprising preparing SEQ ID NO: 29 by coupling: a. SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:33; or b. SEQ ID NO:31, SEQ ID NO:34 and SEQ ID NO:35.