Process for preparing a GLP-1 / glucagon dual agonist

Abstract

Disclosed herein are intermediate preparations for producing glucagon and GLP-1 dual agonist compounds or pharma- ceutically acceptable salts thereof. Also disclosed herein is a process for producing glucagon and GLP-1 dual agonist compounds by coupling two to four intermediate preparations via hybrid solid-liquid phase synthesis.

Description

[Technical field]

[0001] The present invention provides a process for the synthesis of glucagon (GCG) and GLP-1 dual agonist peptides or pharma- ceutically acceptable salts thereof via hybrid solid liquid phase synthesis (hybrid SPPS-LPPS, also referred to herein as HSLPS). [Background technology]

[0002] Over the past few decades, the prevalence of diabetes has been increasing. Type 2 diabetes mellitus ("T2D") is the most common form of diabetes, accounting for approximately 90% of all diabetes cases. T2D is characterized by high blood glucose levels caused by insulin resistance. Uncontrolled diabetes leads to several pathologies that affect the morbidity and mortality of patients. The leading cause of death in diabetic patients is cardiovascular complications. One of the main risk factors for type 2 diabetes is obesity. It has been demonstrated that reduction of body fat accumulation would result in improvement of obesity-related comorbidities, including hyperglycemia and cardiovascular events. Therefore, effective therapies in glucose control and weight loss are needed for better disease management.

[0003] GCG helps maintain blood glucose levels by binding to the GCG receptor on liver cells and causing the liver to release glucose (stored in the form of glycogen) through glycogenolysis. As these stores become depleted, GCG stimulates the liver to synthesize additional glucose through gluconeogenesis. This glucose is released into the bloodstream, preventing the development of hypoglycemia.

[0004] GLP-1 has different biological activities compared to GCG. The actions 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 diabetic patients. Several GLP-1 agonists, including exenatide, liraglutide, lixisenatide, albiglutide, and dulaglutide, have been approved for use in treating T2D in humans. Such GLP-1 agonists have a beneficial effect on body weight, are effective in glycemic control without the risk of hypoglycemia. However, weight loss is moderate due to dose-dependent gastrointestinal side effects.

[0005] GCG and GLP-1 dual agonist peptides that may be useful in the treatment of T2D and obesity are described and claimed in U.S. Patent No. 9,938,335 (B2), in which processes for producing such GCG and GLP-1 dual agonist peptides are described.

[0006] The large-scale preparation of pharma- ceutical elegant GCG and GLP-1 dual agonist peptides presents many technical challenges that can affect overall yield and purity. Methods are also needed to avoid the use of harsh reaction conditions that are incompatible with peptide synthesis.

[0007] WO 2021 / 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 their intermediates are needed to enable pharma-ceutical elegant production with commercially desirable purity and yield. Similarly, methods and stable intermediates for providing GCG and GLP-1 dual agonist peptides efficiently with fewer purification steps are needed. Summary of the Invention [Means for solving the problem]

[0008] The present invention seeks to meet these needs by providing novel intermediates and processes useful for the production of GCG and GLP-1 dual agonist peptide (SEQ ID NO: 1) or pharma- ceutically acceptable salts thereof. The process of the present invention provides intermediates and process reactions that embody a combination of advances, including efficient pathways, while maintaining high quality and purity, reducing resource intensity, and minimizing waste streams.

[0009] In one embodiment, the compound of the formula:

[0010] [ka] [Wherein, the lysine at position 20 (Lys / K) is linked to the epsilon-amino group of the lysine side chain by ([2-(2-aminoethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO-(CH2) 18- A process is provided for preparing a compound of formula (I) which is chemically modified by attachment to COH (SEQ ID NO:1).

[0011] The process of the invention includes an HSLPS method, which uses two to four intermediate preparations to make the compound of SEQ ID NO: 1. As used herein, the term "preparation" refers to a compound, such as a peptide fragment or a fatty acid moiety, used in the synthesis of the compound of SEQ ID NO: 1.

[0012] In one embodiment, the method can include at least the step of coupling three intermediate preparations, such preparations having the structures set forth in SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:5, or pharma- ceutically acceptable salts thereof.

[0013] Alternatively, the method can include at least the step of coupling three intermediate preparations, such preparations having the structures set forth in SEQ ID NO:2, SEQ ID NO:10, and SEQ ID NO:12, or pharma- ceutically acceptable salts thereof.

[0014] Alternatively, the method can include at least the step of coupling three intermediate preparations, such preparations having the structures set forth in SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:12, or pharma- ceutically acceptable salts thereof.

[0015] Alternatively, the method can include at least the step of coupling three intermediate preparations, such preparations having the structures set forth in SEQ ID NO:17, SEQ ID NO:21, and SEQ ID NO:5, or pharma- ceutically acceptable salts thereof.

[0016] Alternatively, the method can include at least the step of coupling three intermediate preparations, such preparations having the structures set forth in SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:12, or pharma- ceutically acceptable salts thereof.

[0017] Alternatively, the method can include at least the step of coupling two intermediate preparations, such preparations having the structures set forth in SEQ ID NO:24 and SEQ ID NO:25, or pharma- ceutically acceptable salts thereof.

[0018] In the above methods, the fatty acid moiety and linker can be attached to one intermediate preparation prior to coupling of the various intermediate preparations (i.e., acylation can be performed prior to complete synthesis).

[0019] Alternatively, the fatty acid moiety can be attached to the peptide after coupling of various intermediate preparations (ie, acylation can be performed after complete synthesis and selective Lys20 deprotection).

[0020] For example, the method can include at least the step of coupling three intermediate preparations, such preparations having the structures set forth in SEQ ID NO:2, SEQ ID NO:7, and SEQ ID NO:5, or pharma- ceutically acceptable salts thereof, followed by coupling with a fatty acid moiety having the structure:

[0021] [ka]

[0022] Alternatively, the method can include at least the step of coupling three intermediate preparations, such preparations having the structures set forth in SEQ ID NO:2, SEQ ID NO:14, and SEQ ID NO:12, or pharma- ceutically acceptable salts thereof, followed by coupling with a fatty acid moiety having the structure of Preparation 30.

[0023] Alternatively, the method can include at least the step of coupling two intermediate preparations, such preparations having the structures set forth in SEQ ID NO:27 and SEQ ID NO:25, or pharma- ceutically acceptable salts thereof, followed by coupling with a fatty acid moiety having the structure of Preparation 30.

[0024] Alternatively, the method can include at least the step of coupling a preparation having the structure set forth in SEQ ID NO: 30, or a pharma- ceutically acceptable salt thereof, followed by coupling with a fatty acid moiety having the structure of preparation 30.

[0025] The above method may also include a step of synthesizing an intermediate preparation prior to the coupling step.

[0026] Thus, in the above methods, intermediate preparations can be chemically or enzymatically coupled to one another to give the compound of SEQ ID NO:1.

[0027] In addition to the methods described above, embodiments of the present invention also include the intermediate preparations themselves (eg, SEQ ID NOs: 2-39), as well as compositions containing them.

[0028] Advantages of the methods herein include several process improvements, such as the generally higher purity and yield of shorter peptide fragments generated initially via solid phase peptide synthesis, i.e., SPPS, via HSLPS.

[0029] The advantages of the method of the present invention include that the efficiency of coupling in SPPS is not only dependent on the actual residues involved in the chemical transformation, but is also influenced by the structure attached to the resin (i.e., solubility / aggregation issues are known for certain sequences, specifically GCG). Shorter fragments allow more flexibility in the coupling pathways of complex amino acid residues, and the fragment structure can be redesigned to address more challenging transformations.

[0030] Advantages of the method of the present invention include improved strategies for controlling impurities during synthesis, which may improve the final impurity profile of the crude peptide and simplify / reduce the chromatographic burden, resulting in cost savings.

[0031] Advantages of the method of the present invention include that the synthesis of short fragments via SPPS allows for shorter wash cycles, reduced reagent amounts, and the use of greener solvents leading to reduced process mass intensity (PMI).

[0032] The advantages of the method of the present invention include that the short fragments significantly reduce the risk of failure that is typical in the construction of long linear molecules, particularly molecules containing GCG-like sequences.

[0033] Advantages of the method of the present invention include that the combination of solution and solid phase synthesis is more suitable for the introduction of new manufacturing platforms such as continuous chemistry and other innovative technologies. Advantages of the method of the present invention include flexibility in the supply chain and logistics of the manufacturing process by using several independent fragments. Advantages of the method of the present invention include that the use of parallel production of fragments allows for a shortened manufacturing cycle due to parallel processing of fragments. Advantages of the method of the present invention include that the current good manufacturing practice (cGMP) solution phase steps can be carried out in standard manufacturing facilities without the need for specialized equipment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] 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 disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used for the synthesis, the preferred methods and materials are described herein.

[0035] Also, the reference to an element by the indefinite article "a" or "an" does not exclude the possibility that a plurality of elements is present, unless the context clearly requires that there is one and only one element. Thus, the indefinite article "a" or "an" normally means "at least one."

[0036] Abbreviations and Definitions Certain abbreviations are defined as follows: "AEEA" refers to 2-[2-(2-amino-ethoxy)-ethoxy]-acetyl; "Aib" refers to α-aminoisobutyric acid; "Alloc" means allyloxycarbonyl; "Boc" refers to tert-butoxycarbonyl; "Bu" refers to butyl; "t-Bu" refers to tert-butyl; "CTC" refers to chlorotrityl chloride; "DCM" refers to dichloromethane; "DIC" refers to diisopropylcarbodiimide; "DMF" refers to dimethylformamide; "DMSO" refers to dimethylsulfoxide; "Dnp" refers to 2,4-dinitrophenyl; "DTT" refers to dithiothreitol; "Fmoc" refers to fluorenylmethyloxycarbonyl chloride; and "hr" refers to hours. In the above example, "HFIP" refers to hexafluoroisopropanol, "IPA" refers to isopropyl, "IPAc" refers to isopropyl acetate, "min" refers to minutes, "Me" refers to methyl, "MTBE" refers to methyl tert-butyl ether, "MTT" refers to 4-methyltrityl, "oxyma" refers to ethyl cyanohydroxyiminoacetate, "OtBu" refers to tert-butyl ester, "PG" refers to a protecting group, "Pip" refers to piperidine, "PyBOP" refers to (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate), "SPPS" refers to solid phase peptide synthesis, "TFA" refers to trifluoroacetic acid, "TIPS" refers to triisopropylsilane, and "Trt" refers to trityl.

[0037] As used herein, "about" means within a statistically significant range of a value(s), such as, for example, a stated concentration, length, molecular weight, pH, sequence identity, time frame, temperature, or volume. Such values ​​or ranges may be within an order of magnitude, typically within 20%, more typically within 10%, and even more typically within 5% of a given value or range. The allowable variation encompassed by "about" will depend on the particular system under study and can be readily appreciated by one of ordinary skill in the art.

[0038] As used herein, the term "protected" means that a protecting group is attached at the indicated position. Those skilled in the art will recognize that a variety of protecting groups are well known and that alternative protecting groups may be suitable for a particular process.

[0039] The present disclosure provides and therefore encompasses novel intermediate compounds (herein referred to as "preparations" or "intermediate preparations"), such as peptide fragments and fatty acid moieties useful for the synthesis of the compound of SEQ ID NO:1, as well as methods for synthesizing the compound of SEQ ID NO:1 or its pharma- ceutically acceptable salts. The intermediate preparations herein can be prepared by a variety of techniques known in the art. For example, a method using standard solid-phase peptide synthesis of two or more intermediate preparations followed by their coupling in solution phase is illustrated in the examples below. The specific synthetic steps for each of the described routes or schemes may be combined in different methods to prepare the compounds described herein. The reagents and starting materials are readily available to one of ordinary skill in the art.

[0040] method Standard solid phase peptide synthesis of intermediate preparation: The intermediate peptide fragments herein can be made via any number of standard peptide synthesis methods known in the art, specifically SPPS. SPPS construction is carried out using standard Fmoc peptide chemistry techniques with sequential couplings on an automated peptide synthesizer such as a Gyros Protein Technologies Symphony X synthesizer. The method of SPPS is well known in the art and does not need to be exhaustively described here. For a general discussion, see "Fmoc Solid Phase Peptide Synthesis: A Practical Approach" (Chan & White ed., Oxford University Press 2000), and Merrifield (1963) J. Am. Chem. Soc. 85:2149-2154.

[0041] For deprotection, the resin is swelled in DMF and then deprotected using 20% ​​Pip / DMF (3x30 min). Subsequent Fmoc deprotections use successive 20% Pip / DMF treatments, with additional treatment steps used for more difficult deprotections.

[0042] After deprotection, the resin is washed with DMF. Preactivation of amino acids uses DIC / Oxyma DMF solution for 30 min at room temperature. Coupling of activated amino acids to resin-bound peptides is performed for specific times for each individual amino acid. After coupling is complete, DMF washes are performed after each coupling to remove excess reagents.

[0043] To isolate the final product, the resin-bound product is washed with DCM to remove DMF. The resin is washed with IPA to remove DCM, then with MTBE, and the product is then dried under vacuum at 40° C. The resin-bound product is stored refrigerated (−20° C.).

[0044] Soft cleavage procedure for peptides that maintain the protecting groups: The peptide on resin intermediate is treated with a solution of 30% HFIP in DCM. The spent resin is filtered off and then washed with DCM. The combined filtrates are poured into 7-10 volumes of cold (0 °C) MTBE. The suspension is aged at 0 °C for 30 min, the resulting precipitate is centrifuged, and the clear solution is decanted. The residue is suspended in an equal volume of MTBE, and the resulting suspension is centrifuged and decanted again. After decanting, the clear MTBE solution of precipitated peptide is dried in vacuum at 40 °C overnight.

[0045] An alternative soft cleavage process is as follows: The peptide is soft cleaved from the resin with a 1-5% TFA / DCM cocktail. The resin is swelled with DCM (10 volumes, 1 × 20 min) and drained. 1-5% TFA / DCM (10 volumes) is added to the pre-swollen resin and the suspension is stirred at room temperature for 10 h. The solution is filtered and the filtrate is treated with pyridine (equimolar to the TFA added). The resin is treated two more times with 1-5% TFA / DCM (10 volumes), the filtrates are combined and each time treated with pyridine (equimolar to the TFA). The filtrate is concentrated under reduced pressure and the resulting residue is dissolved in DMF and precipitated from cold (0 °C) water (10 volumes relative to DMF). The precipitate is filtered, washed with additional water (4-5 volumes relative to DMF) and dried in vacuum at 40 °C overnight.

[0046] Hard cleavage procedure to remove protecting groups: The peptide is cleaved from the resin with an acidic cocktail containing TFA / H2O / TIPS / DTT in the ratios (0.93v / 0.04v / 0.03v / 0.03w). The resin is swelled in DCM (4-5vol, 3x30min) and drained. The cleavage cocktail (4-5vol) is added to the pre-swollen resin and the suspension is stirred for 2h at room temperature. The solution is filtered, then the resin is washed with a small amount of DCM and mixed with the cleavage solution. The resulting solution is poured into 7-10 volumes of cold (0°C) MTBE. The suspension is aged for 30min at 0°C, the resulting precipitate is centrifuged and the clear solution is decanted. The residue is suspended in the same volume of MTBE and the resulting suspension is centrifuged and decanted again. After decanting, the clear MTBE solution of precipitated peptide is dried in vacuum at 40°C overnight.

[0047] Hybrid solid-liquid phase synthesis The intermediate peptide fragments prepared via SPPS as described above can be combined in solution phase to obtain the dual agonist of SEQ ID NO:1. Methods of HSLPS are well known in the art and need not be exhaustively described herein. For general discussion, see U.S. Patent Application Publication No. 2011 / 0046349, as well as 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 4 th ed. 2016), and Zhang et al. (2008) Org. Process Res. Dev. 12:101-110.

[0048] Briefly, HSLPS involves the synthesis of independent intermediate peptide fragments in solid phase and their coupling in solution phase.

[0049] As applied herein, one method of making a compound of SEQ ID NO:1 includes at least the step of coupling the following three intermediate fragments or preparations, where such preparations have the structures set forth in SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:5. In some examples, the fragments can be coupled in the order SEQ ID NO:2-SEQ ID NO:3-SEQ ID NO:5 (i.e., from the C-terminus to the N-terminus). In other examples, the fragments can be joined in a different order using a strategy that employs appropriate protecting groups.

[0050] A typical coupling protocol is to combine approximately equimolar amounts of the two fragments in a polar solvent such as DMSO. The acid-bearing fragment can be activated using stoichiometric amounts of PYBOP and Hunig's base or related activation systems. After a suitable reaction time, DEA is used for FMOC deprotection. Water is then added to induce precipitation of the resulting peptide coupling product, which is filtered, isolated, and dried.

[0051] Another method for making the compound of SEQ ID NO:1 includes at least the step of coupling the following three intermediate preparations, which preparations have the structures set forth in SEQ ID NO:2, SEQ ID NO:10, and SEQ ID NO:12. In some examples, the fragments are coupled in the order SEQ ID NO:2-SEQ ID NO:10-SEQ ID NO:12 (i.e., from the C-terminus to the N-terminus). In other examples, the fragments can be joined in a different order using a strategy that employs appropriate protecting groups.

[0052] Another method for making the compound of SEQ ID NO:1 includes at least the step of coupling the following three intermediate preparations, where such intermediates have the structures set forth in SEQ ID NOs:17, 18, and 12. In some examples, the fragments are coupled in the order SEQ ID NO:17-SEQ ID NO:18-SEQ ID NO:12 (i.e., from the C-terminus to the N-terminus). In other examples, the fragments can be joined in a different order using a strategy that employs appropriate protecting groups.

[0053] Another method for making the compound of SEQ ID NO:1 includes at least the step of coupling the following three intermediate preparations, which preparations have the structures set forth in SEQ ID NO:17, SEQ ID NO:21, and SEQ ID NO:5. In some examples, the fragments are coupled in the order SEQ ID NO:17-SEQ ID NO:21-SEQ ID NO:5 (i.e., from the C-terminus to the N-terminus). In other examples, the fragments can be joined in a different order using a strategy that employs appropriate protecting groups.

[0054] Another method for making the compound of SEQ ID NO:1 includes at least the step of coupling the following three intermediate preparations, which preparations have the structures set forth in SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:12. In some examples, the fragments are coupled in the order SEQ ID NO:36-SEQ ID NO:37-SEQ ID NO:12 (i.e., from the C-terminus to the N-terminus). In other examples, the fragments can be joined in a different order using a strategy that employs appropriate protecting groups.

[0055] Another method for producing the compound of SEQ ID NO:1 includes at least the step of coupling the following two intermediate preparations, which preparations have the structures set forth in SEQ ID NO:24 and SEQ ID NO:25:

[0056] Alternatively, another method for making compounds of SEQ ID NO:1 uses the same cleavage as above, but instead couples all amino acid fragments of the backbone first, then introduces the fatty acid side moiety as the last chemical transformation, followed by global deprotection, where, for example, the corresponding PG can be placed at Lys20, which can be selectively removed in the presence of other PGs (e.g., Boc, Dnp, tBu, and / or Trt).

[0057] In some examples, the method of making a compound of SEQ ID NO:1 includes at least the step of coupling the following intermediate preparations, such preparations having the structures set forth in SEQ ID NO:2, SEQ ID NO:7, and SEQ ID NO:5, as well as the structure of Preparation 30. In some examples, the fragments are coupled in the order SEQ ID NO:2-SEQ ID NO:7-SEQ ID NO:5 (i.e., from the C-terminus to the N-terminus), followed by coupling with Preparation 30. In other examples, the fragments can be joined in a different order using a strategy that employs appropriate protecting groups.

[0058] Another method of making the compound of SEQ ID NO:1 includes at least the step of coupling the following three intermediate preparations, which preparations have the structures set forth in SEQ ID NO:2, SEQ ID NO:14, and SEQ ID NO:12, as well as Preparation 30. In some examples, the fragments are coupled in the order SEQ ID NO:2-SEQ ID NO:14-SEQ ID NO:12 (i.e., from the C-terminus to the N-terminus), followed by coupling with Preparation 30. In other examples, the fragments can be joined in a different order using a strategy that employs appropriate protecting groups.

[0059] Another method for making the compound of SEQ ID NO:1 includes at least the step of coupling the following two intermediate preparations, which preparations have the structures set forth in SEQ ID NO:27 and SEQ ID NO:25, and the structure of Preparation 30. In some examples, the fragments are coupled in the order SEQ ID NO:27-SEQ ID NO:25, followed by coupling with Preparation 30. In other examples, the fragments can be joined in a different order using a strategy that employs appropriate protecting groups.

[0060] In some examples, the method for producing the compound of SEQ ID NO:1 includes at least the step of coupling the following intermediate preparation, which has the structure set forth in SEQ ID NO:30 and the structure of intermediate preparation 30:

[0061] For efficient synthesis of intermediates (acylated peptide fragments) of SEQ ID NO:3, SEQ ID NO:10, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, and SEQ ID NO:37, the following are synthesized with fatty acid side chains:

[0062] [ka] The Fmoc-L-Lys-OH amino acid is attached to the fatty side chain.

[0063] [ka]

[0064] To improve the purity and efficiency of SPPS, the following tetramers and pentamers (SEQ ID NOs: 31, 32, 33, 34, and 35) can be used to prepare intermediate preparations 4, 11, 24, and 28 (SEQ ID NOs: 5, 12, 25, and 29), and the following structures can be synthesized using amino acid building blocks via SPPS or solution phase synthesis:

[0065] [ka]

[0066] 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.

[0067] Those skilled in the art will recognize that a variety of protecting groups are well known and alternative protecting groups may be used for the amino acids, for example, instead of Dnp, trityl or Boc may be present on the histidine in Boc-H(Dnp)-Aib-Q(Trt)-G) (SEQ ID NO:31).

[0068] The compounds of SEQ ID NO:1 herein can be used in a number of therapeutic applications, for example in methods of treating obesity, type 2 diabetes, nonalcoholic fatty liver disease (NAFLD), and / or nonalcoholic steatohepatitis (NASH) in an individual.

[0069] Compounds of SEQ ID NO:1 may be prepared as described in Hybrid Synthesis Schemes 1-10 shown below. The examples are provided for purposes of illustration and not limitation. EXAMPLES

[0070] Example 1 (Scheme 1):

[0071] [ka]

[0072] Example 2 (Scheme 2):

[0073] [ka]

[0074] Example 3 (Scheme 3):

[0075] [ka]

[0076] Example 4 (Scheme 4):

[0077] [ka]

[0078] Example 5 (Scheme 5):

[0079] [ka]

[0080] Example 6 (Scheme 6):

[0081] [ka]

[0082] Example 7 (Scheme 7):

[0083] [ka]

[0084] Example 8 (Scheme 8):

[0085] [ka]

[0086] Example 9 (Scheme 9):

[0087] [ka]

[0088] Example 30 (Scheme 10)

[0089] [ka]

[0090] In Example 1 (Scheme 1), Preparation 1 (SEQ ID NO:2), Preparation 2 (SEQ ID NO:3), and Preparation 4 (SEQ ID NO:5) are prepared by SPPS, Preparation 3 (SEQ ID NO:4), Preparation 5 (SEQ ID NO:6) are prepared by LPPS, and Compound 1 (SEQ ID NO:1) is prepared via deprotection of Preparation 5 (SEQ ID NO:6).

[0091] In Example 2 (Scheme 2), Preparation 1 (SEQ ID NO:2), Preparation 6 (SEQ ID NO:7), and Preparation 4 (SEQ ID NO:5) are prepared by SPPS, Preparation 7 (SEQ ID NO:8), Preparation 8 (SEQ ID NO:9) are prepared by LPPS, and Preparation 30 is coupled to the Lys side chain of Preparation 8, followed by a deprotection step to give Compound 1 (SEQ ID NO:1).

[0092] In Example 3 (Scheme 3), Preparation 1 (SEQ ID NO:2), Preparation 9 (SEQ ID NO:10), and Preparation 11 (SEQ ID NO:12) are prepared by SPPS, Preparation 10 (SEQ ID NO:11), Preparation 12 (SEQ ID NO:13) are prepared by LPPS, and Compound 1 (SEQ ID NO:1) is prepared via deprotection of Preparation 12 (SEQ ID NO:13).

[0093] In Example 4 (Scheme 4), Preparation 1 (SEQ ID NO:2), Preparation 13 (SEQ ID NO:14), and Preparation 11 (SEQ ID NO:12) are prepared by SPPS, Preparation 14 (SEQ ID NO:15), Preparation 15 (SEQ ID NO:16) are prepared by LPPS, and Preparation 30 is coupled to the Lys side chain of Preparation 15, followed by a deprotection step to give Compound 1 (SEQ ID NO:1).

[0094] In Example 5 (Scheme 5), Preparation 16 (SEQ ID NO: 17), Preparation 17 (SEQ ID NO: 18), and Preparation 11 (SEQ ID NO: 12) are prepared by SPPS, Preparation 18 (SEQ ID NO: 19), Preparation 19 (SEQ ID NO: 20) are prepared by LPPS, and Compound 1 (SEQ ID NO: 1) is prepared via deprotection of Preparation 19 (SEQ ID NO: 20).

[0095] In Example 6 (Scheme 6), Preparation 16 (SEQ ID NO: 17), Preparation 20 (SEQ ID NO: 21), and Preparation 4 (SEQ ID NO: 5) are prepared by SPPS, Preparation 21 (SEQ ID NO: 22), Preparation 22 (SEQ ID NO: 23) are prepared by LPPS, and Compound 1 (SEQ ID NO: 1) is prepared via deprotection of Preparation 22 (SEQ ID NO: 23).

[0096] In Example 7 (Scheme 7), Preparation 23 (SEQ ID NO:24), Preparation 24 (SEQ ID NO:25) are prepared by SPPS, and Preparation 25 (SEQ ID NO:26) is prepared by LPPS, followed by deprotection to give Compound 1 (SEQ ID NO:1).

[0097] In Example 8 (Scheme 8), Preparation 24 (SEQ ID NO:25), Preparation 26 (SEQ ID NO:27) are prepared by SPPS, Preparation 27 (SEQ ID NO:28) is prepared by LPPS, and Preparation 30 is coupled to the Lys side chain of Preparation 27, followed by deprotection to give Compound 1 (SEQ ID NO:1).

[0098] In Example 9 (Scheme 9), Preparation 28 (SEQ ID NO: 29) is prepared by SPPS and undergoes soft deprotection to remove MTT to give Preparation 29 (SEQ ID NO: 30), and Preparation 30 is coupled to the Lys side chain of Preparation 29, followed by a deprotection step to give Compound 1 (SEQ ID NO: 1).

[0099] In Example 30 (Scheme 10), Preparation 37 (SEQ ID NO:36), Preparation 38 (SEQ ID NO:37), and Preparation 11 (SEQ ID NO:12) are prepared by SPPS, Preparation 39 (SEQ ID NO:38) and Preparation 40 (SEQ ID NO:39) are prepared by LPPS, and Compound 1 (SEQ ID NO:1) is prepared via deprotection of Preparation 40 (SEQ ID NO:39).

[0100] General steps: Swelling of the resin: The resin (0.500 mmol) is charged into a reactor and allowed to swell with DMF (3 x 10 mL x 20 min).

[0101] Post-Fmoc Deprotection Washing: After deprotection, wash the resin with DMF (5 x 10 mL x 2 min).

[0102] Post-coupling wash: After coupling, wash the resin with DMF (5 x 10 mL x 2 min).

[0103] Washing and drying of the resin: After the last coupling or deprotection, wash the resin with DMF (5 x 10 mL x 2 min), followed by DCM (5 x 10 mL x 2 min) and drain dry under N2 atmosphere until constant weight.

[0104] Example 10: Synthesis of Preparation 1 (SEQ ID NO:2) by Solid Phase Peptide Synthesis

[0105] [ka]

[0106] Preparation 1 (SEQ ID NO:2) or a pharma-ceutically acceptable salt thereof is synthesized by standard SPPS using Sieber amide resin (0.75 mmol / g loading ratio) under the conditions described below.

[0107] [Table 1]

[0108] Cutting from resin: Add 5% TFA / DCM (10 volumes) to the resin and agitate the reactor for 30 minutes. Drain the reactor and wash the resin with DCM (2 x 5 volumes). Add the filtrate to pre-chilled MTBE:heptane (1:1, 10 volumes relative to the cleavage solution) and then centrifuge (3000 rpm x 10 minutes). Discard the supernatant and add fresh cold MTBE:heptane (5 volumes) and centrifuge the mixture (3000 rpm x 5 minutes). Discard the supernatant and repeat the process once more with fresh MTBE:heptane. Discard the supernatant and place the resulting material in a vacuum oven at 34°C for 14 hours to give Preparation 1. Observed mass: 515.8 [M+H].

[0109] Example 11: Synthesis of Preparation 2 (SEQ ID NO:3) by Solid Phase Peptide Synthesis

[0110] [ka]

[0111] Preparation 2 (SEQ ID NO:3) or a pharma-ceutically acceptable salt thereof is synthesized by standard SPPS using Fmoc-Gly-CTC resin (0.84 mmol / g loading ratio) in the conditions described below.

[0112] [Table 2]

[0113] Cutting from resin: The peptide intermediate is soft cleaved from the resin using a 20% HFIP / DCM solution. The peptide intermediate on the resin is swelled with DCM (2 x 15 min x 10 volumes). The resin is then treated with 20% HFIP / DCM (5 x 20 min x 5 volumes) and the filtrates are combined. The filtrate is concentrated to 1 / 3 volume at 30°C and this volume is replaced with chloroform (x3). The solution is then concentrated under reduced pressure to give a viscous residue which is then added dropwise to n-heptane cooled to -15°C under vigorous stirring. The precipitate is filtered off and the residue is washed with n-heptane (x3). The resulting solid is then dried under vacuum for at least 18 hours. Actual mass: 3306.98

[0114] Example 12: Synthesis of Preparation 4 (SEQ ID NO:5) by Solid Phase Peptide Synthesis

[0115] [ka]

[0116] Preparation 4 (SEQ ID NO:5) or a pharma-ceutically acceptable salt thereof is synthesized by standard SPPS using Fmoc-L-Asp(OtBu)-CTC resin (0.67 mmol / g loading ratio) under the conditions described below.

[0117] [Table 3]

[0118] Cutting from resin: The peptide intermediate is soft cleaved from the resin using a 20% HFIP / DCM solution. The peptide intermediate on the resin is swelled with DCM (2 x 15 min x 10 volumes). The resin is then treated with 20% HFIP / DCM (5 x 20 min x 5 volumes) and the filtrates are combined. The filtrate is concentrated to 1 / 3 volume at 30°C and this volume is replaced with chloroform (x3). The solution is then concentrated under reduced pressure to give a viscous residue which is then added dropwise to n-heptane cooled to -15°C under vigorous stirring. The precipitate is filtered off and the residue is washed with n-heptane (x3). The resulting solid is then dried under vacuum for at least 18 hours. Actual mass: 2802.51

[0119] Example 13: Synthesis of Preparation 6 (SEQ ID NO:7) by Solid Phase Peptide Synthesis

[0120] [ka]

[0121] Preparation 6 (SEQ ID NO: 7) or a pharma-ceutically acceptable salt thereof is synthesized by standard SPPS using Fmoc-Gly-CTC resin (0.84 mmol / g loading ratio) in the conditions described below.

[0122] [Table 4]

[0123] Cutting from resin: The peptide intermediate is soft cleaved from the resin using a 20% HFIP / DCM solution. The peptide intermediate on the resin is swelled with DCM (2 x 15 min x 10 volumes). The resin is then treated with 20% HFIP / DCM (5 x 20 min x 5 volumes) and the filtrates are combined. The filtrate is concentrated to 1 / 3 volume at 30°C and this volume is replaced with chloroform (x3). The solution is then concentrated under reduced pressure to give a viscous residue which is then added dropwise to n-heptane cooled to -15°C under vigorous stirring. The precipitate is filtered off and the residue is washed with n-heptane (x3). The resulting solid is then dried under vacuum for at least 18 hours. Actual mass: 2535.41

[0124] Example 14: Synthesis of Preparation 11 (SEQ ID NO:12) by Solid Phase Peptide Synthesis

[0125] [ka]

[0126] Preparation 11 (SEQ ID NO: 12) or a pharma-ceutically acceptable salt thereof is synthesized by standard SPPS using Fmoc-L-Asp(OtBu)-CTC resin (0.67 mmol / g loading ratio) under the conditions described below.

[0127] [Table 5] * Boc-L-His(Boc)-OH can be used in cycle 8 to prepare Boc-His(Boc)-containing fragments.

[0128] Cutting from resin: To the resin in a fritted reactor equipped with an overhead stirrer, add DCM (10 vol) and agitate the reactor for 10 minutes to swell the resin. Drain the reactor and add 1% TFA / DCM (10 vol) to the resin. Agitate the reactor for 10 minutes, drain the reactor, and collect the filtrate. Add fresh 1% TFA / DCM (10 vol) to the reactor and agitate the reactor for 10 minutes. Add pyridine (1:1 equivalent to TFA added) to the filtrate. Drain the reactor and combine with the previous filtrate. Add fresh 1% TFA / DCM (10 vol) to the reactor again and agitate the reactor for 10 minutes. Add additional pyridine (1:1 equivalent to TFA added) to the filtrate. Drain the reactor and combine the filtrate. Add pyridine (1:1 equivalent to TFA added) to the filtrate. The reactor containing the spent resin is washed with DCM (2 x 10 volumes) and combined with the previous filtrate. The filtrate is concentrated under reduced pressure. The resulting residue is dissolved in a minimum of DMF and added dropwise to cold water (15 volumes relative to the DMF added to dissolve the residue) to precipitate the peptide. The suspension is filtered through a fritted funnel and the solid is rinsed with cold water (2 x 10 volumes). The resulting solid is placed in a vacuum oven at 34 °C for 14 hours to obtain the Preparation 11 fragment.

[0129] Boc-His(Boc)-containing: M / Z measured values ​​= 1642.8911 and 1542.8396 [M-Boc].

[0130] Containing Boc-His(Dnp): ​​M / Z measured value = 1708.8410.

[0131] Example 15: Synthesis of Preparation 16 (SEQ ID NO:17) by Solid Phase Peptide Synthesis

[0132] [ka]

[0133] Preparation 16 (SEQ ID NO: 17) or a pharma- ceutically acceptable salt thereof is synthesized by standard SPPS using Sieber amide resin (0.75 mmol / g loading ratio) under the conditions described below.

[0134] [Table 6]

[0135] Cutting from resin: The resin (3 g) is loaded into a fritted reactor equipped with an overhead stirrer. The resin is suspended in DCM (28.5 mL) and TFA (1.5 mL) is added to the suspension. The resulting suspension is mixed for 30 min and the reactor is drained. The resin bed is washed with DCM (20 mL) and the filtrate is poured into pre-chilled MTBE:heptane (1:1, 300 mL). The suspension is centrifuged (3000 rpm x 10 min) and the supernatant is discarded. Fresh pre-chilled MTBE:heptane (1:1, 300 mL) is added and the suspension is centrifuged again (3000 rpm x 5 min). The supernatant is discarded and the wash is repeated once more with fresh MTBE:heptane (1:1, 300 mL). After centrifugation, the supernatant is discarded and the resulting solid is placed in a vacuum oven at 34 °C for 14 h to give Preparation 16. Actual mass: 1699.9829.

[0136] Example 16: Synthesis of Preparation 17 (SEQ ID NO:18) by Solid Phase Peptide Synthesis Preparation 17 (SEQ ID NO: 18), or a pharma- ceutically acceptable salt thereof, is synthesized by standard SPPS using the conditions described below.

[0137] [ka]

[0138] Preparation 17 (SEQ ID NO: 18) or a pharma-ceutically acceptable salt thereof is synthesized by standard SPPS using Fmoc-L-Glu(OtBu)-CTC resin (0.655 mmol / g loading ratio) under the conditions described below.

[0139] [Table 7]

[0140] Cutting from resin: To the resin in a fritted reactor equipped with an overhead stirrer, add DCM (10 vol) and agitate the reactor for 10 minutes to swell the resin. Drain the reactor and add 1% TFA / DCM (10 vol) to the resin. Agitate the reactor for 10 minutes, drain the reactor, and collect the filtrate. Add fresh 1% TFA / DCM (10 vol) to the reactor and agitate the reactor for 10 minutes. Add pyridine (1:1 equivalent to TFA added) to the filtrate. Drain the reactor and combine with the previous filtrate. Add fresh 1% TFA / DCM (10 vol) to the reactor again and agitate the reactor for 10 minutes. Add additional pyridine (1:1 equivalent to TFA added) to the filtrate. Drain the reactor and combine the filtrate. Add pyridine (1:1 equivalent to TFA added) to the filtrate. The reactor containing the spent resin is washed with DCM (2 x 10 volumes) and combined with the previous filtrate. The filtrate is concentrated under reduced pressure. The resulting residue is dissolved in a minimum of DMF and added dropwise to cold water (15 volumes relative to the DMF added to dissolve the residue) to precipitate the peptide. The suspension is filtered through a fritted funnel and the solid is rinsed with cold water (2 x 10 volumes). The resulting solid is placed in a vacuum oven at 34°C for 14 hours to give Preparation 17. Observed mass: 1609.1 [M+H]

[0141] Example 17: Synthesis of Preparation 32 (SEQ ID NO:31) by Solid Phase Peptide Synthesis

[0142] [ka]

[0143] Preparation 32 (SEQ ID NO: 31) or a pharma-ceutically acceptable salt thereof is synthesized by standard SPPS using Fmoc-Gly-CTC resin (1.04 mmol / g loading ratio) in the conditions described below.

[0144] [Table 8]

[0145] Cutting from resin: The peptide is soft cleaved from the resin using a 30% HFIP / DCM solution. The pre-swollen resin containing the Preparation 33 peptide is treated with 30% HFIP / DCM (10 volumes) and agitated for 2 h. The suspension is filtered and the resin cake is washed with DCM (4-5 volumes). The filtrate is concentrated under reduced pressure and then stripped three times with ACN to obtain the Preparation 32 peptide as a dry foam.

[0146] Example 18: Synthesis of Preparation 33 (SEQ ID NO:32) by Solid Phase Peptide Synthesis

[0147] [ka]

[0148] Preparation 33 (SEQ ID NO: 32) or a pharma-ceutically acceptable salt thereof is synthesized by standard SPPS using Fmoc-L-Ser(tBu)-CTC resin (0.71 mmol / g loading ratio) under the conditions described below.

[0149] [Table 9]

[0150] Cutting from resin: The peptide is soft cleaved from the resin using a 30% HFIP / DCM solution. The pre-swollen resin containing the Preparation 33 peptide is treated with 30% HFIP / DCM (10 volumes) and agitated for 2 h. The suspension is filtered and the resin cake is washed with DCM (4-5 volumes). The filtrate is concentrated under reduced pressure and then stripped three times with ACN to obtain the Preparation 33 peptide as a dry foam.

[0151] Example 19: Synthesis of Preparation 34 (SEQ ID NO:33) by Solid Phase Peptide Synthesis

[0152] [ka]

[0153] Preparation 34 (SEQ ID NO: 33) or a pharma-ceutically acceptable salt thereof is synthesized by standard SPPS using Fmoc-L-Lys(Boc)-CTC resin (0.92 mmol / g loading ratio) under the conditions described below.

[0154] [Table 10] Table 10

[0155] Cutting from resin: The pre-swollen resin containing Preparation 34 peptide is treated with 30% HFIP / DCM (10 volumes) and stirred for 2 h. The suspension is filtered and the resin cake is washed with DCM (4-5 volumes). The filtrate is concentrated under reduced pressure and then stripped three times with ACN to obtain Preparation 34 peptide as a dry foam.

[0156] Example 20: Synthesis of Preparation 35 (SEQ ID NO:34) by Solid Phase Peptide Synthesis

[0157] [ka]

[0158] Preparation 35 (SEQ ID NO: 34) or a pharma-ceutically acceptable salt thereof is synthesized by standard SPPS using Fmoc-L-Asp(tBu)-CTC resin (0.67 mmol / g loading ratio) under the conditions described below.

[0159] [Table 11]

[0160] Cutting from resin: The pre-swollen resin containing Preparation 35 peptide is treated with 30% HFIP / DCM (10 volumes) and stirred for 2 h. The suspension is filtered and the resin cake is washed with DCM (4-5 volumes). The filtrate is concentrated under reduced pressure and then stripped three times with ACN to obtain Preparation 35 peptide as a dry foam.

[0161] Example 21: Synthesis of Compound 31 Preparation 31 or ((52S)-52-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-25-(tert-butoxycarbonyl)-2,2-dimethyl-4,23,28,37,46-pentaoxo-3,32,35,41,44-pentaoxa-24,29,38,47-tetraazatripentacontan-53-oic acid) is prepared as described in WO 2020 / 159949.

[0162] Example 22: Synthesis of Preparation 9 (SEQ ID NO:10) by Solid Phase Peptide Synthesis

[0163] [ka]

[0164] Preparation 9 (SEQ ID NO: 10), or a pharma- ceutically acceptable salt thereof, is synthesized by standard SPPS using H-Gly-CTC resin in the conditions described below.

[0165] [Table 12-1]

[0166] [Table 12-2]

[0167] Cutting from resin: The peptide intermediate is soft cleaved from the resin using a 20% HFIP / DCM solution. The peptide intermediate on the resin is swelled with DCM (2 x 15 min x 10 volumes). The resin is then treated with 20% HFIP / DCM (5 x 20 min x 5 volumes) and the filtrates are combined. The filtrate is concentrated to 1 / 3 volume at 30°C and this volume is replaced with chloroform (x3). The solution is then concentrated under reduced pressure to give a viscous residue which is then added dropwise to n-heptane cooled to -15°C under vigorous stirring. The precipitate is filtered off and the residue is washed with n-heptane (x3). The resulting solid is then dried under vacuum for at least 18 hours. Actual mass: 4400.64

[0168] Example 23: Synthesis of Preparation 13 (SEQ ID NO:14) by Solid Phase Peptide Synthesis

[0169] [ka]

[0170] Preparation 13 (SEQ ID NO: 14), or a pharma- ceutically acceptable salt thereof, is synthesized by standard SPPS using H-Gly-CTC resin in the conditions described below.

[0171] [Table 13-1] Table 13

[0172] [Table 13-2]

[0173] Cutting from resin: The peptide intermediate is soft cleaved from the resin using a 20% HFIP / DCM solution. The peptide intermediate on the resin is swelled with DCM (2 x 15 min x 10 volumes). The resin is then treated with 20% HFIP / DCM (5 x 20 min x 5 volumes) and the filtrates are combined. The filtrate is concentrated to 1 / 3 volume at 30°C and this volume is replaced with chloroform (x3). The solution is then concentrated under reduced pressure to give a viscous residue which is then added dropwise to n-heptane cooled to -15°C under vigorous stirring. The precipitate is filtered off and the residue is washed with n-heptane (x3). The resulting solid is then dried under vacuum for at least 18 hours. Actual mass: 3629.07

[0174] Example 24: Synthesis of Preparation 20 (SEQ ID NO:21) by Solid Phase Peptide Synthesis

[0175] [ka]

[0176] Preparation 20 (SEQ ID NO: 21) or a pharma-ceutically acceptable salt thereof is synthesized by standard SPPS using Fmoc-L-Glu(OtBu)-CTC resin (0.920 mmol / g loading ratio) under the conditions described below.

[0177] [Table 14] Table 14

[0178] Cutting from resin: To the resin in a fritted reactor equipped with an overhead stirrer, add DCM (10 vol) and agitate the reactor for 10 minutes to swell the resin. Drain the reactor and add 1% TFA / DCM (10 vol) to the resin. Agitate the reactor for 10 minutes, drain the reactor, and collect the filtrate. Add fresh 1% TFA / DCM (10 vol) to the reactor and agitate the reactor for 10 minutes. Add pyridine (1:1 equivalent to TFA added) to the filtrate. Drain the reactor and combine with the previous filtrate. Add fresh 1% TFA / DCM (10 vol) to the reactor again and agitate the reactor for 10 minutes. Add additional pyridine (1:1 equivalent to TFA added) to the filtrate. Drain the reactor and combine the filtrate. Add pyridine (1:1 equivalent to TFA added) to the filtrate. The reactor containing the spent resin is washed with DCM (2 x 10 volumes) and combined with the previous filtrate. The filtrate is concentrated under reduced pressure. The resulting residue is dissolved in a minimum of DMF and added dropwise to cold water (15 volumes relative to the DMF added to dissolve the residue) to precipitate the peptide. The suspension is filtered through a fritted funnel and the solid is rinsed with cold water (2 x 10 volumes). The resulting solid is placed in a vacuum oven at 34 °C for 14 hours to obtain the fragment of preparation 20. Actual mass: 2121.2953

[0179] Example 25: Synthesis of Preparation 36 (SEQ ID NO:35) by Solid Phase Peptide Synthesis

[0180] [ka]

[0181] Preparation 36 (SEQ ID NO:35) or a pharma- ceutically acceptable salt thereof is synthesized by standard SPPS using CTC resin (0.671 mmol / g loading ratio) in the conditions described below.

[0182] [Table 15]

[0183] Cutting from resin: Treat the pre-swollen resin containing Preparation 36 peptide with 30% HFIP / DCM (10 volumes) and agitate for 2 h. Filter the suspension and wash the resin cake with DCM (4-5 volumes). Concentrate the filtrate under reduced pressure and then strip with ACN three times to obtain Preparation 36 peptide as a dry foam.

[0184] Example 26: Synthesis of Preparation 23 (SEQ ID NO:24) by Solid Phase Peptide Synthesis

[0185] [ka]

[0186] Preparation 23 (SEQ ID NO:24) or a pharma- ceutically acceptable salt thereof is synthesized by standard SPPS using Sieber amide resin (0.75 mmol / g loading ratio) under the conditions described below.

[0187] [Table 16]

[0188] Cutting from resin: A sample of the resin was taken for hard cleavage to confirm the success of the assembly. The resin (200 mg) was diluted with 2.5 mL of cleavage cocktail (2.32 mL TFA, 63 mg DTT, 0.063 mL TIPS, 0.063 mL H x O) is added and the suspension is mixed for 2.5 hours. The resin is filtered off, rinsed with 1 mL of TFA, and the filtrate is poured into pre-chilled MTBE (17.5 mL). The suspension is aged at 0°C for 30 minutes, then centrifuged (3000 rpm x 10 minutes). The supernatant is discarded and the solid is washed with fresh MTBE (18 mL), centrifuged (3000 rpm x 5 minutes), and decanted. This is repeated once more with fresh MTBE, and the resulting solid is vacuum dried for 14 hours. Actual mass: 1300.7 [M+2 / 2]

[0189] Example 27: Synthesis of Preparation 24 (SEQ ID NO:25) by Solid Phase Peptide Synthesis

[0190] [ka]

[0191] Preparation 24 (SEQ ID NO:25), or a pharma-ceutically acceptable salt thereof, is synthesized by standard SPPS using Fmoc-Ala-CTC resin, followed by soft cleavage from the resin.

[0192] Example 28: Synthesis of Preparation 26 (SEQ ID NO:27) by Solid Phase Peptide Synthesis

[0193] [ka]

[0194] Preparation 26 (SEQ ID NO:27), or a pharma- ceutically acceptable salt thereof, is synthesized by standard SPPS using Sieber amide resin (0.75 mmol / g loading ratio) under the conditions described below.

[0195] [Table 17]

[0196] Cleavage from resin: A sample of the resin was taken for hard cleavage to confirm the success of the assembly. The resin (200 mg) was diluted with 2.5 mL of cleavage cocktail (2.32 mL TFA, 63 mg DTT, 0.063 mL TIPS, 0.063 mL H). x 0) was added and the suspension was mixed for 2.5 hours. The resin was filtered off, rinsed with 1 mL of TFA, and the filtrate was poured into pre-chilled MTBE (17.5 mL). The suspension was aged at 0°C for 30 minutes and then centrifuged (3000 rpm x 10 minutes). The supernatant was discarded and the solid was washed with fresh MTBE (18 mL), centrifuged (3000 rpm x 5 minutes), and decanted. This was repeated once more with fresh MTBE, and the resulting solid was vacuum dried for 14 hours. Actual mass: 1940.7

[0197] Example 29: Synthesis of Preparation 29 (SEQ ID NO:30) by Solid Phase Peptide Synthesis

[0198] [ka]

[0199] Preparation 29 (SEQ ID NO: 30) or a pharma- ceutically acceptable salt thereof is synthesized by standard SPPS using Sieber resin (0.75 mmol / g loading ratio) under the conditions described below.

[0200] [Table 18-1]

[0201] [Table 18-2]

[0202] Soft cleavage of peptide from resin: A fritted reactor is charged with the resin (20 g) followed by DCM (10 volumes). The resulting suspension is stirred with an overhead stirrer for 20 minutes to swell the resin. The reactor is drained and 20 volumes of a 5% TFA / DCM solution is charged to the reactor and the resulting suspension is stirred for 30 minutes. The reactor is drained into a round-bottom flask and the resin cake is washed with DCM (3 x 5 volumes). Pyridine (equimolar to the TFA added) is added to the filtrate and then the filtrate is concentrated under reduced pressure. The resulting red-orange residue is taken up in 25 mL of DMF and added dropwise to cold water (200 mL) to precipitate the peptide. The round-bottom flask is rinsed with 2 x 5 mL portions of DMF and added dropwise to the water / peptide suspension. The resulting suspension is placed in the refrigerator for 30 minutes and then filtered through a fritted funnel. The solid is washed with additional cold water and then placed in a vacuum oven at 35°C overnight to give 15.9 g of the desired peptide. Actual mass: 5444.1861[M]; 5344.1325[M-Boc]

[0203] Example 31: Synthesis of Preparation 37 (SEQ ID NO:36) by Solid Phase Peptide Synthesis

[0204] [ka]

[0205] Preparation 37 (SEQ ID NO:36) or a pharma- ceutically acceptable salt thereof is synthesized by standard SPPS using Sieber amide resin (0.75 mmol / g loading ratio) under the conditions described below.

[0206] [Table 19]

[0207] Cutting from resin: The resin (2 g) was loaded into a fritted 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 min and the reactor was drained. The resin bed was washed with DCM (10 mL) and the filtrate was poured into pre-chilled MTBE:heptane (1:1, 200 mL). The suspension was centrifuged (3000 rpm x 10 min) and the supernatant was discarded. Fresh pre-chilled MTBE:heptane (1:1, 200 mL) was added and the suspension was centrifuged again (3000 rpm x 5 min). The supernatant was discarded and the wash was repeated one more time 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 h to give Preparation 37. Actual mass: 1885.097

[0208] Example 32: Synthesis of Preparation 38 (SEQ ID NO:37) by Solid Phase Peptide Synthesis

[0209] [ka]

[0210] Preparation 38 (SEQ ID NO: 37) or a pharma- ceutically acceptable salt thereof is synthesized by standard SPPS using Preparation 31-CTC resin (0.3574 mmol / g loading ratio) in the conditions described below.

[0211] [Table 20]

[0212] Cutting from resin: Load a 20 mL scintillation vial with the resin (1 g). Suspend the resin in DCM (7 mL) and add HFIP (3 mL) to the suspension. Cap the vial and place on a rotating wheel for 2 h. Filter the resin and wash the resin cake with DCM (10 mL). Concentrate the filtrate under reduced pressure and take up the resulting residue in MeCN and concentrate to dryness (3 times) to give Preparation 38 as a sticky yellow solid. Observed mass: 3029.8636

[0213] Example 33: Synthesis of Compound 1 using Scheme 5 Solution phase coupling of Preparation 16 and Preparation 17 forms Preparation 18: To a 2.5 wt% solution of Preparation 16 (97 mg) in DMF is added a 2.5 wt% solution of Preparation 17 (1.3 eq.) in DMF. The resulting solutions are mixed and to the mixture is added 2.5 wt% PyAOP (8.7 eq.) followed by a solution of DIPEA (2.1 eq.) in MeCN. The resulting reaction mixture is stirred at room temperature for 2 hours, after which DEA ​​(11.5 eq.) is added. The mixture is stirred at room temperature for 1 hour. Observed mass: 1559.8 [M+3 / 3]

[0214] Solution phase coupling of Preparation 18 and Preparation 11 forms Preparation 19: To a 5 wt% solution of Preparation 18 (40 mg) in DMF is added a 5 wt% solution of Preparation 11 (1.6 eq.). The solutions are mixed and a 5 wt% solution of PyAOP (3.0 eq.) in MeCN is added to the solution, followed by a 5 wt% solution of DIPEA (8 eq.) in DMF. The resulting reaction mixture is stirred at room temperature for 2 h 40 min, after which 1.0 mL of a 17% NaCl / 0.5% NaHCO3 solution is added and stirred for 5 min. Then 1.0 mL of cold deionized water is added and the resulting mixture is stirred at 0° C. for 1 h. The white precipitate is then filtered through a fritted funnel and washed with deionized water (2 mL). The resulting solid is dried overnight at 30° C. in a vacuum oven to give the desired material as an off-white solid.

[0215] Global deprotection of preparation 19 results in the formation of compound 1 (SEQ ID NO:1): To the solid obtained above, add TFA (0.424 mL), TIPS (11.5 μL), water (11.5 μL), and DTT (11.5 mg). The resulting mixture is mixed on a rotating wheel for 2 hours and then poured into 11 mL of cold MTBE. The resulting precipitate is centrifuged at 3000 rpm for 3 minutes and washed twice with MTBE, centrifuging each time. The solid is allowed to dry at room temperature. Measured mass: 1521.9 [M+3 / 3]

[0216] Example 34: Synthesis of Compound 1 using Scheme 6 Liquid phase coupling of Preparation 16 and Preparation 20 forms Preparation 21: A 250 mL round bottom flask equipped with a magnetic stir bar is charged with Preparation 16 (1.26 g, 1.02 eq.). 5 mL portions of DMF / THF (85 / 15, 20 mL) are added to the flask. The material is stirred for approximately 5 minutes to completely dissolve the material. Meanwhile, a 20 mL scintillation vial is charged with Preparation 20 (2.10 g, 1.00 eq.) and PyAOP (450 mg, 1.56 eq.) and the material is dissolved in DMF / THF (85 / 15, 10 mL) and then added to the round bottom flask. The vial is rinsed with 2×5 mL portions of DMF / THF (85 / 15). DIPEA (0.67 mL, 7.00 eq.) is added to the reaction mixture and the resulting reaction mixture is stirred at room temperature. After 18 hours, DEA (0.57 mL, 10 eq.) is added and the resulting solution is stirred for 1 hour. After 1 h, the reaction mixture is added dropwise to pre-chilled 17% NaCl / 0.5% NaHCO3 aqueous solution (400 mL) to precipitate the peptide. The suspension is filtered and the solid is washed with 2 x 50 mL cold deionized water. The solid is dried overnight at 34 °C in a vacuum oven. Measured mass: 3581.2002

[0217] Solution phase coupling of Preparation 21 and Preparation 4 forms Preparation 22: To a solution of Preparation 21 (522 mg, 1.05 eq.) in DMF / THF (85 / 15, 8 mL) is added a solution of Preparation 4 (containing His(Dnp)) (385 mg, 1.02 eq.) and PyAOP (66 mg, 1.53 eq.) in DMF / THF (85 / 15, 3 mL). The solution is mixed for 5 minutes to dissolve these substances, after which DIPEA (0.044 mL, 3.00 eq.) is added to the reaction mixture. The resulting reaction mixture is allowed to stir at room temperature for 4 hours, after which the peptide is precipitated by dropwise addition to 110 mL of pre-chilled 17% NaCl / 0.5% NaHCO3 aqueous solution. The suspension is then filtered through a fritted funnel and the solid is washed with 2 x 25 mL of deionized water. The solid is then dried in a vacuum oven at 34 °C for 14 hours. Measured mass: 6365.6991

[0218] Global deprotection of preparation 22 results in the formation of compound 1 (SEQ ID NO:1): To the solid obtained above, add TFA (0.424 mL), TIPS (11.5 μL), water (11.5 μL), and DTT (11.5 mg). The resulting mixture is mixed on a rotating wheel for 2 hours and then poured into 11 mL of cold MTBE. The resulting precipitate is centrifuged at 3000 rpm for 3 minutes and washed twice with MTBE, centrifuging each time. The solid is allowed to dry at room temperature. Measured mass: 1521.9 [M+3 / 3]

[0219] Example 35: Synthesis of Compound 1 using Scheme 9 Solution phase coupling of Preparation 29 and Preparation 30 results in the formation of Compound 1 (SEQ ID NO:1): Coupling of Preparation 30 with Preparation 29: A reaction vessel equipped with a magnetic stir bar is charged with Preparation 29 (2 g) and DMF (15 mL). The resulting mixture is mixed until the peptide is completely dissolved (approximately 10 min). A solution of Preparation 30 (2.0 eq.) and PyBOP (2.0 eq.) in DMF (5 mL) is prepared and then added to the peptide solution. The reaction mixture is charged with DIPEA (4.0 eq.) and the resulting reaction mixture is allowed to stir for 18 h. The reaction mixture is then added dropwise to 200 mL of pre-chilled 17% NaCl / 0.5% NaHCO3 aqueous solution to precipitate the peptide. The resulting suspension is aged in the refrigerator for 30 min and then filtered through a fritted funnel. The solid is washed with additional cold water and then dried overnight in a vacuum oven to give the desired peptide (2.16 g). Actual mass: 6299.7459[M]; 6199.6908[M-Boc]

[0220] Global deprotection: To the protected peptide (1 g), 10 volumes of cleavage cocktail (92.5% TFA:2.5% TIPS:2.5% DTT:2.5% HO) are added. The resulting mixture is allowed to mix for 2 h and then added dropwise into pre-chilled MTBE (7 volumes) to precipitate the peptide. The suspension is centrifuged (3000 rpm x 10 min) and then the supernatant is discarded. The solid is then suspended in fresh MTBE (7 volumes) and centrifuged again (3000 rpm x 5 min). The supernatant is discarded and the process is repeated once more with fresh MTBE (7 volumes). After centrifugation, the supernatant is discarded and the resulting solid is dried in a vacuum oven overnight to obtain the crude peptide. Observed mass: 4560.2610.

[0221] Example 36: Synthesis of Compound 1 using Scheme 10 Solution phase coupling of Preparation 37 and Preparation 38 forms Preparation 39: To a solution of Preparation 37 (54 mg, 1.18 eq.) in DMF / THF (85 / 15, 0.5 mL), add a solution of Preparation 38 (73 mg, 1.00 eq.) and PyAOP (26 mg, 2.10 eq.) in DMF / THF (85 / 15, 3 mL). The mixture is stirred for about 5 minutes to dissolve the solids, after which DIPEA (0.03 mL, 7.89 eq.) is added and the resulting reaction mixture is stirred. After 4 hours, DEA (0.03 mL, 11.3 eq.) is added and the resulting mixture is stirred at room temperature for 1 hour. The reaction mixture is then added dropwise to a pre-cooled 17% NaCl / 0.5% NaHCO3 aqueous solution (35 mL) to precipitate the peptide. The suspension is filtered through a fritted funnel and the solid is washed with 2 x 10 mL of deionized water. Dry the solid in a vacuum oven at 34° C. for 14 hours to give Preparation 39 in 49% crude yield. Found mass: 4674.8656

[0222] Solution phase coupling of Preparation 39 and Preparation 11 forms Preparation 40: Preparation 39 and Preparation 11 are coupled in solution phase using the coupling conditions described in Scheme 5, second coupling step (Example 33).

[0223] Global deprotection of preparation 40 results in the formation of compound 1 (SEQ ID NO:1): Global deprotection of preparation 40 is carried out using the conditions described in Example 33.

[0224] Example 37: Synthesis of Preparation 11 using Tetramer Preparation 32 and Preparation 33 Fmoc-Asp(OtBu)-CTC (0.500 mmol, 0.67 mmol / g) resin is loaded into a solid phase reactor, then swollen with DMF (3 x 10 mL x 20 min), then deprotected with 20% piperidine / DMF (3 x 10 mL x 30 min). To the preactivation vessel, 4 mL of a 0.375 M solution of Preparation 33 (Fmoc-T(tBu)-FT(tBu)-S(tBu)-OH) in DMF is added, followed by 2 mL of a 0.750 M solution of Oxyma in DMF and 2.5 mL of a 0.660 M solution of DIC in DMF. The resulting solution is mixed with N2 bubbling for 30 min, then transferred to the reactor containing the resin and allowed to couple for 12 h. The reactor is drained, then the resin is washed with DMF (5 x 10 mL x 2 min). Fmoc is removed with 20% piperidine / DMF (3 x 10 mL x 30 min), then the resin is washed with DMF (5 x 10 mL x 2 min). To the preactivation vessel, 4 mL of a 0.375 M solution of Preparation 32 (Boc-His(dnp)-Aib-Q(Trt)-G-OH) in DMF is added, followed by 2 mL of a 0.750 M solution of Oxyma in DMF and 2.5 mL of a 0.660 M solution of DIC in DMF. The resulting solution is mixed with N2 bubbling for 30 min, then transferred to the reactor containing the resin and allowed to couple for 12 h. The reactor is then drained and the resin is washed with DMF (5 x 10 mL x 2 min), followed by DCM (5 x 10 mL x 2 min), then the effluent is dried under an N2 sweep for 8 h. A sample of the resin is then hard cleaved (see hard cleavage procedure) to confirm successful assembly. Measured mass: 1143.3.

[0225] array The following sequences are mentioned in this disclosure and are provided below for reference:

[0226] SEQ ID NO:1

[0227] [ka] H-Aib-QGTFTSDYSKYLDEKKAK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO-(CH2) 18 -CO2H)EFVEWLLEGGPSSG-NH2

[0228] SEQ ID NO:2

[0229] [ka]

[0230] SEQ ID NO:3

[0231] [ka]

[0232] SEQ ID NO:4

[0233] [ka]

[0234] SEQ ID NO:5

[0235] [ka]

[0236] SEQ ID NO:6

[0237] [ka]

[0238] SEQ ID NO:7

[0239] [ka]

[0240] SEQ ID NO:8

[0241] [ka]

[0242] SEQ ID NO:9

[0243] [ka]

[0244] SEQ ID NO:10

[0245] [ka]

[0246] SEQ ID NO:11

[0247] [ka]

[0248] SEQ ID NO:12

[0249] [ka]

[0250] SEQ ID NO:13

[0251] [ka]

[0252] SEQ ID NO:14

[0253] [ka]

[0254] SEQ ID NO:15

[0255] [ka]

[0256] SEQ ID NO:16

[0257] [ka]

[0258] SEQ ID NO:17

[0259] [ka]

[0260] SEQ ID NO:18

[0261] [ka]

[0262] SEQ ID NO:19

[0263] [ka]

[0264] SEQ ID NO:20

[0265] [ka]

[0266] SEQ ID NO:21

[0267] [ka]

[0268] SEQ ID NO:22

[0269] [ka]

[0270] SEQ ID NO:23

[0271] [ka]

[0272] SEQ ID NO:24

[0273] [ka]

[0274] SEQ ID NO:25

[0275] [ka]

[0276] SEQ ID NO:26

[0277] [ka]

[0278] SEQ ID NO:27

[0279] [ka]

[0280] SEQ ID NO:28

[0281] [ka]

[0282] SEQ ID NO:29

[0283] [ka]

[0284] SEQ ID NO:30

[0285] [ka]

[0286] SEQ ID NO:31 Boc-H(Dnp)-Aib-Q(Trt)-G

[0287] SEQ ID NO:32 Fmoc-T(tBu)-FT-(tBu)-S(tBu)

[0288] SEQ ID NO:33 Fmoc-D(tBu)-Y(tBu)-S(tBu)-K(Boc)

[0289] SEQ ID NO:34 Fmoc-T(tBu)-FT(tBu)-S(tBu)-D(tBu)

[0290] SEQ ID NO:35 Fmoc-Y(t-Bu)-S(t-Bu)-K(Boc)-Y(t-Bu)

[0291] SEQ ID NO:36

[0292] [ka]

[0293] SEQ ID NO:37

[0294] [ka]

[0295] SEQ ID NO:38

[0296] [ka]

[0297] SEQ ID NO:39

[0298] [ka]

Claims

1. 1. A method of making a compound of SEQ ID NO:1 or a pharma- ceutically acceptable salt thereof, comprising: 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 Preparation 30, and j. A method comprising coupling an intermediate preparation selected from the group consisting of SEQ ID NO:30 and Preparation 30.

2. 2. The method of claim 1, wherein the method comprises coupling SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:

12.

3. 2. The method of claim 1, wherein the method comprises coupling SEQ ID NO:17, SEQ ID NO:21, and SEQ ID NO:

5.

4. 2. The method of claim 1, wherein the method comprises coupling SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:

12.

5. 2. The method of claim 1, wherein the method comprises coupling SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:

5.

6. 2. The method of claim 1, wherein the method comprises coupling SEQ ID NO:2, SEQ ID NO:10, and SEQ ID NO:

12.

7. 2. The method of claim 1, wherein the method comprises coupling SEQ ID NO:24 and SEQ ID NO:

25.

8. 2. The method of claim 1, wherein the method comprises coupling SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:5, and Preparation 30.

9. 2. The method of claim 1, wherein the method comprises coupling SEQ ID NO:2, SEQ ID NO:14, SEQ ID NO:12, and preparation 30.

10. 2. The method of claim 1, wherein the method comprises coupling SEQ ID NO:27, SEQ ID NO:25, and Preparation 30.

11. The method of claim 1, wherein the method comprises coupling SEQ ID NO: 30 and preparation 30.

12. The method further comprising:

3. The method of claim 2, comprising synthesizing each of SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:12 by solid phase peptide synthesis and coupling them in solution phase.

13. The method further comprising:

4. The method of claim 3, comprising synthesizing each of SEQ ID NO:17, SEQ ID NO:21, and SEQ ID NO:5 by solid phase peptide synthesis and coupling them in solution phase.

14. The method further comprising:

5. The method of claim 4, comprising synthesizing each of SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:12 by solid phase peptide synthesis and coupling them in solution phase.

15. The method further comprising:

6. The method of claim 5, comprising synthesizing each of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:5 by solid phase peptide synthesis and coupling them in solution phase.

16. The method further comprising:

7. The method of claim 6, comprising synthesizing each of SEQ ID NO:2, SEQ ID NO:10, and SEQ ID NO:12 by solid phase peptide synthesis and coupling them in solution phase.

17. The method further comprising:

8. The method of claim 7, comprising synthesizing each of SEQ ID NO:24 and SEQ ID NO:25 by solid phase peptide synthesis and coupling them in solution phase.

18. The method further comprising:

9. The method of claim 8, comprising synthesizing each of SEQ ID NO:2, SEQ ID NO:7, and SEQ ID NO:5 by solid phase peptide synthesis, coupling them in liquid phase, and subsequently coupling them with preparation 30 in liquid phase.

19. The method further comprising:

10. The method of claim 9, comprising synthesizing each of SEQ ID NO:2, SEQ ID NO:14, and SEQ ID NO:12 by solid phase peptide synthesis, coupling them in liquid phase, and subsequently coupling them with preparation 30 in liquid phase.

20. The method further comprising:

11. The method of claim 10, comprising synthesizing each of SEQ ID NO:27 and SEQ ID NO:25 by solid phase peptide synthesis, coupling them in liquid phase, and subsequently coupling them with preparation 30 in liquid phase.

21. The method further comprising:

12. The method of claim 11, comprising synthesizing SEQ ID NO:30 by solid phase peptide synthesis and coupling it with preparation 30 in liquid phase.

22. An intermediate preparation 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 a pharma- ceutically acceptable salt thereof.

23. An intermediate preparation 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 a pharma- ceutically acceptable salt thereof.

24. a. SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33, or b. The method of claim 1, comprising preparing SEQ ID NO:5 by coupling SEQ ID NO:31, SEQ ID NO:34, and SEQ ID NO:

35.

25. 2. The method of claim 1, comprising preparing SEQ ID NO:12 by coupling SEQ ID NO:31 and SEQ ID NO:

34.

26. a. SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33, or b. The method of claim 1, comprising preparing SEQ ID NO:25 by coupling SEQ ID NO:31, SEQ ID NO:34, and SEQ ID NO:

35.

27. a. SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33, or b. The method of claim 1, comprising preparing SEQ ID NO:29 by coupling SEQ ID NO:31, SEQ ID NO:34, and SEQ ID NO:35.

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