Process for preparing GLP-1 / glucagon dual agonist

Abstract

To provide glucagon (Gcg) and GLP-1 dual agonist peptides useful in the treatment of type 2 diabetes, obesity, nonalcoholic fatty liver disease (NAFLD), and / or nonalcoholic steatohepatitis (NASH).SOLUTION: Provided is a compound having the following formula.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a glucagon (Gcg) and GLP-1 dual agonist peptide or its

[0010] The present invention provides a method for making a pharmaceutically acceptable salt of

[0002] The prevalence of diabetes has been increasing over the past few decades. Type 2 diabetes mellitus (T2D) affects It is the most common form of diabetes, accounting for approximately 90% of all diabetes cases. Uncontrolled diabetes is characterized by high blood sugar levels caused by resistance. It leads to several pathologies that affect morbidity and mortality. It is the main cause of death in diabetic patients. The cause is cardiovascular complications. One of the main risk factors for type 2 diabetes is obesity. Most T2D patients (approximately 90%) are overweight or obese. It has been demonstrated to improve obesity-related comorbidities, including diabetes and cardiovascular events. Therefore, effective therapies in glucose control and weight loss are needed for better disease management. is required.

[0003] Gcg binds to the Gcg receptor on hepatocytes and is transported to the liver through glycogenolysis (glycogen Helps maintain blood sugar levels by releasing glucose (stored in the form of As these stores are depleted, Gcg stimulates the liver to produce additional glucose through gluconeogenesis. This glucose is released into the bloodstream and helps prevent hypoglycemia. do.

[0004] GLP-1 has different biological activities compared to Gcg. The actions of GLP-1 include: These include stimulation of insulin synthesis and secretion, inhibition of Gcg secretion, and inhibition of food intake. GLP-1 has been shown to reduce hyperglycemia in diabetic patients. Includes senatide, liraglutide, lixisenatide, albiglutide, and dulaglutide Several GLP-1 agonists are approved for use in the treatment of T2D in humans. Such GLP-1 agonists have beneficial effects on body weight and reduce the risk of hypoglycemia. It is non-toxic and effective in controlling glycemia. However, it is known to cause dose-dependent gastrointestinal side effects. As a result of its action, weight loss is moderate.

[0005] Gcg and GLP-1 dual agonists may be useful in the treatment of T2D and obesity The peptide is described and claimed in U.S. Patent No. 9,938,335 B2. Methods for producing Gcg and GLP-1 dual agonist peptides such as are.

[0006] However, improvements for the production of Gcg and GLP-1 dual agonist peptides There remains a need for a method that provides a commercially desirable purity. Similarly, Gcg and GLP-1 dual agonist peptides have a combination of benefits including and stabilized compounds to provide peptides with fewer or easier purification steps. Therefore, efficient and environmentally friendly "green" methods are needed. Large-scale preparation of g and GLP-1 dual agonist peptides has been shown to improve overall yield and purity These present many technical challenges that can affect the success of peptide synthesis. There is also a need for methods to avoid the use of reactive conditions.

[0007] The present invention relates to a Gcg and GLP-1 dual agonist peptide (SEQ ID NO: 1), or By providing a novel method useful for preparing pharmaceutically acceptable salts thereof, The improved manufacturing method of the present invention aims to meet this need. Combinations of advances, including efficient paths with fewer steps while simultaneously maintaining Importantly, improved methods and chemical reactions are provided. The compound reduces resource strength.

[0008] The improved methods described herein involve the use of dual Gcg and GLP-1 agonists. The present invention provides a variety of compounds useful for producing peptides.

[0009] In particular, the following formula: [ka] [wherein the lysine at position 20 (Lys / K) is in contact with the epsilon-amino group of the lysine side chain ([ 2-(2-aminoethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO-(C H2) 18 modified by bonding with COH (SEQ ID NO: 1) A method for preparing The method comprises: (i) the following formula: [ka] wherein PG1 is a base-stable side chain protecting group; Thr at position 5 is optionally protected by PG1; PG2 is an ivDde, Dde, or Alloc side chain protecting group (SEQ ID NO: 2). solid phase synthesis of the compound of formula (I); (ii) selectively deprotecting the lysine to give Lys-NH2 (SEQ ID NO: 5) t B uO-C 20 -γGlu( t Coupling with Bu)-AEEA-AEEA-OH selectively acylating Lys at position 20 (SEQ ID NO: 7) by (iii) cleaving the compound from the solid support and removing the base-stable side chain protecting groups; , (iv) purifying the compound (SEQ ID NO: 1).

[0010] Peptides in which side chains (e.g., fatty acid side chains) are built stepwise by individual couplings Conventional preparations of hydroxybenzoates produce significant amounts of addition and deletion by-products. This results in an unfavorable purity profile that makes the purification of peptide compounds difficult. When the EEA spacer is part of a side chain constructed by conventional methods, the yield is low. The following is a typical example.

[0011] Selective deprotection of Lys at position 20 and subsequent acylation afforded the hydroxyl group as an intact fragment. t Bu O.C. 20 -γGlu( t Derivatives coupled to the (Bu)-AEEA-AEEA-OH side chains With deprotected 1-34Lys-20-NH2 peptide (SEQ ID NO: 4) on the lipid backbone This represents a novelty of on-resin large fragment coupling. The acylation of peptides or proteins is an efficient method for producing compounds in high yields. Acylation is achieved with over 99% selectivity and minimal impurities. This occurs at the lysine at the desired position. Selective deprotection and subsequent coupling affords This results in a favorable impurity profile for the acylation reaction. The acylation process facilitates the purification and isolation of the desired acylated peptide product; This results in higher yields and purity.

[0012] Selective deprotection of Lys at position 20 allows the ivDde, Dde, or Alloc side chain at position 20 to be This is facilitated by the use of protecting groups and base-stable side chain protecting groups at other positions. De, Dde, or Alloc side chain protecting groups are removed, but base-stable side chain protecting groups (PG Deprotection conditions are chosen such that 1) remains intact.

[0013] A variety of base-stable protecting groups are known in the art and can be used in the methods of the present invention. In one embodiment of the present invention, the base-stable side chain protecting groups PG1 used in the synthesis of the compounds (a) in the case of Trp and Lys, by tert-butyloxycarbonyl (Boc) (b) tert-butyl esters (O t Bu) (c) tert-butyl in the case of Ser, Thr, and Tyr ( t Bu) and ( d) In the case of Gln, it is triphenylmethyl (Trityl) (Trt), and (e) H In the case of is, it is Boc(Boc) or Boc(Dnp).

[0014] In a preferred embodiment of the method of the present invention, the side chain protecting group at Lys at position 20 is ivD It is de.

[0015] In an alternative embodiment of the method of the present invention, the side chain protecting group of Lys at position 20 is Dde. .

[0016] Dde is a protecting group that is stable to most conventional bases and therefore is suitable for Fmoc ivDde is a derivative of Dde and is also stable to removal conditions. A further advantage of ivDde is that it is stable to removal conditions due to its steric hindrance. The main difference is that Dde and ivDde have a reduced tendency to migrate to free Lys residues. It is generally removed by hydrazinolysis.

[0017] Preferably, when PG2 is ivDde or Dde, Lys at position 20 is a compound It is selectively deprotected by contact with a solution containing hydrazine hydrate.

[0018] More preferably, the solution is 1% to 15% w / w in DMF, NMP, NBP, or DMSO. / w of hydrazine hydrate.

[0019] Even more preferably, the solution contains 8% w / w hydrazine hydrate in DMF. .

[0020] In an alternative embodiment of the method of the present invention, the side chain protecting group of Lys at position 20 is selected from Alloc be.

[0021] Alloc is a base-labile protecting group. It is generally used to The side chain protection is removed by palladium catalysis in the presence of a scavenger. The use of groups includes Boc / Bn and Fmoc / t Compatible with the Bu strategy and the presence of acylating agents Tandem elimination-acylation reaction when palladium-catalyzed amino deblocking is carried out under This approach prevents the formation of diketopiperazines (DKPs).

[0022] Preferably, when the side chain protecting group at Lys at position 20 is Alloc, the side chain protecting group at L at position 20 is Alloc. ys can be selectively synthesized by contacting the compound with a palladium catalyst in the presence of a scavenger. It is deprotected.

[0023] More preferably, in the presence of H3N·BH3, Me2NH·BH3, or PhSiH3 By contacting the compound with Pd(PPh3)4, the All of Lys at the specified position was The oc side chain protecting groups are removed.

[0024] The deprotected (20) compound is washed, deswelled, isolated, dried, and packaged. The deprotected (20-position) compound is reswelled before coupling with the side chain.

[0025] In a preferred embodiment of the method of the present invention, PG1 is Boc in the case of Trp and Lys. and O in the case of Asp and Glu. t Bu, and Ser, Thr, and Tyr teeth t Bu, Trt for Gln, and Boc for His. and PG2 is ivDde, and the solid phase synthesis of the compound of step (i) (SEQ ID NO: 3) The procedure is carried out on an Fmoc amide resin solid support, followed by Fmoc deprotection of the amide resin and the following reaction: Sequential Coupling: Fmoc-L-Gly-OH, Fmoc-L-Ser( t Bu)-OH, Fmoc-L -Ser( t Bu)-OH, Fmoc-L-Pro-OH, Fmoc-L-Gly-OH , Fmoc-L-Gly-OH, Fmoc-L-Glu(O t Bu)-OH, Fmoc- L-Leu-OH, Fmoc-L-Leu-OH, Fmoc-L-Trp(Boc)-O H, Fmoc-L-Glu(O t Bu)-OH, Fmoc-L-Val-OH, Fmoc -L-Phe-OH, Fmoc-L-Glu(O t Bu)-OH, Fmoc-Lys(i vDde)-OH, Fmoc-L-Ala-OH, Fmoc-L-Lys(Boc)-O H, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Glu(O t Bu)-OH , Fmoc-L-Asp(O t Bu)-OH, Fmoc-L-Leu-OH, Fmoc- L-Tyr( t Bu)-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L- Ser( t Bu)-OH, Fmoc-L-Tyr( t Bu)-OH, Fmoc-L-As p(O t Bu)-OH, Fmoc-L-Ser( t Bu)-OH, Fmoc-L-Thr ( t Bu)-OH, Fmoc-L-Phe-OH, Fmoc-Gly-Thr(ψ Me, Me Pro)-OH, Fmoc-L-Gln(Trt)-OH, Fmoc-Aib-OH and Boc-L-His(Boc)-OH.

[0026] In an alternative embodiment of the method of the present invention, PG1 is Boc(Dnp) in the case of His. and the solid phase synthesis of the compound in step (i) is carried out as described above.

[0027] The solid phase synthesis of the compounds is carried out on an Fmoc amide resin solid support, and the first step is Fmoc deprotection of the amide resin followed by sequential coupling of Fmoc amino acids to the peptide The glycine-threonine pseudoproline dipeptide is a carboxyl group at positions 4 and 5. For coupling, the individual Fmoc-L-Gly and Fmoc-L-Thr amino acids In these embodiments, the Thr residue at position 5 is used instead. Therefore, the specific Thr residue is reversibly protected as a P There is no need to protect with G1. The reaction is A significant benefit is realized in terms of progression to completion. Coupling of L-Gly and Fmoc-L-Thr amino acids resulted in the formation of a Thr5 deletion. This results in high levels of peptide impurities.

[0028] In an alternative preferred embodiment of the method of the present invention, PG1 is is Boc, and in the cases of Asp and Glu, t Bu, Ser, Thr, Tyr In the case of t Bu, Trt in the case of Gln, and Boc(D np), PG2 is ivDde, and the compound of step (i) (SEQ ID NO: 4) Solid-phase synthesis is carried out on an Fmoc amide resin solid support, and the Fmoc deprotection and and the following sequential couplings: Fmoc-L-Gly-OH, Fmoc-L-Ser( t Bu)-OH, Fmoc-L -Ser( t Bu)-OH, Fmoc-L-Pro-OH, Fmoc-L-Gly-OH , Fmoc-L-Gly-OH, Fmoc-L-Glu(O t Bu)-OH, Fmoc- L-Leu-OH, Fmoc-L-Leu-OH, Fmoc-L-Trp(Boc)-O H, Fmoc-L-Glu(O t Bu)-OH, Fmoc-L-Val-OH, Fmoc -L-Phe-OH, Fmoc-L-Glu(O t Bu)-OH, Fmoc-Lys(i vDde)-OH, Fmoc-L-Ala-OH, Fmoc-L-Lys(Boc)-O H, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Glu(O t Bu)-OH , Fmoc-L-Asp(O t Bu)-OH, Fmoc-L-Leu-OH, Fmoc- L-Tyr( t Bu)-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L- Ser( t Bu)-OH, Fmoc-L-Tyr( t Bu)-OH, Fmoc-L-As p(O t Bu)-OH, Fmoc-L-Ser( t Bu)-OH, Fmoc-L-Thr ( t Bu)-OH, Fmoc-L-Phe-OH, and Boc-His(Dnp)-Ai b-Gln(Trt)-Gly-Thr( t Bu)-OH.

[0029] The solid phase synthesis of the compounds is carried out on an Fmoc amide resin solid support, and the first step is Fmoc deprotection of the amide resin followed by sequential coupling of Fmoc amino acids to the peptide The ring is Boc-His(Dnp)-Aib-Gln(Trt)-Gly-Thr. ( t The (Bu)-OH pentamer (SEQ ID NO: 14) is synthesized as a single fragment from the H2N-6-34 intermediate. This preferred embodiment is coupled to Phe6 (SEQ ID NO: 10) of A significant benefit realized through this is improved purity due to minimization of histidine racemization. be.

[0030] The compound of SEQ ID NO:4 has a nucleotide sequence at lysine 20 as described herein. The resulting compound has the following formula (SEQ ID NO: 18): [ka]

[0031] The compound of SEQ ID NO:18 may be, as an intact fragment as described herein, t B uO-C 20 -γGlu( t Coupling with the (Bu)-AEEA-AEEA-OH side chain The resulting compound has the following formula (SEQ ID NO: 19): [ka]

[0032] In a further alternative preferred embodiment of the method of the present invention, PG1 comprises (a) Trp and L (b) Boc in the case of Asp and Glu; t Bu, (c ) For Ser, Thr, and Tyr t Bu, and (d) Trt in the case of Gln. (e) In the case of His, it is Boc(Dnp), PG2 is ivDde, and ste The solid phase synthesis of compound (i) (SEQ ID NO: 4) was carried out on an Fmoc amide resin solid support. This is followed by Fmoc deprotection of the amide resin and the following sequential couplings: Fmoc-L-Gly-OH, Fmoc-L-Ser( t Bu)-OH, Fmoc-L -Ser( t Bu)-OH, Fmoc-L-Pro-OH, Fmoc-L-Gly-OH , Fmoc-L-Gly-OH, Fmoc-L-Glu(O t Bu)-OH, Fmoc- L-Leu-OH, Fmoc-L-Leu-OH, Fmoc-L-Trp(Boc)-O H, Fmoc-L-Glu(O t Bu)-OH, Fmoc-L-Val-OH, Fmoc -L-Phe-OH, Fmoc-L-Glu(O t Bu)-OH, Fmoc-Lys(i vDde)-OH, Fmoc-L-Ala-OH, Fmoc-L-Lys(Boc)-O H, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Glu(O t Bu)-OH , Fmoc-L-Asp(O t Bu)-OH, Fmoc-L-Leu-OH, Fmoc- L-Tyr( t Bu)-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L- Ser( t Bu)-OH, Fmoc-L-Tyr( t Bu)-OH, Fmoc-L-As p(O t Bu)-OH, Fmoc-L-Ser( t Bu)-OH, Fmoc-L-Thr ( t Bu)-OH, Fmoc-L-Phe-OH, Fmoc-L-Thr( t Bu)-O H, and Boc-His(Dnp)-Aib-Gln(Trt)-Gly-OH.

[0033] The solid phase synthesis of the compounds is carried out on an Fmoc amide resin solid support, and the first step is Fmoc deprotection of the amide resin followed by sequential coupling of Fmoc amino acids to the peptide Boc-His(Dnp)-Aib-Gln(Trt)-Gly-OH tetramer The 2HN-5-34 intermediate (SEQ ID NO: 16) was synthesized as a single fragment from Thr5 (SEQ ID NO: 17). No. 12). The benefit of this is improved purity due to minimization of histidine racemization.

[0034] The compound of SEQ ID NO:4 has a nucleotide sequence at lysine 20 as described herein. The resulting compound has the formula SEQ ID NO:18.

[0035] The compound of SEQ ID NO:18 may be, as an intact fragment as described herein, t B uO-C 20 -γGlu( t Coupling with the (Bu)-AEEA-AEEA-OH side chain The resulting compound has the formula of SEQ ID NO:19.

[0036] In a preferred embodiment of the method of the present invention, the resin solid support is an Fmoc amide resin solid support. and the solid phase synthesis includes Fmoc deprotection of the resin.

[0037] More preferably, the Fmoc amide resin solid support is Sieber resin.

[0038] In one embodiment of the present invention, step (iii) comprises the cleaved and deprotected compound The pH of the solution is adjusted to 7.0-8.0 and stirred for 1-24 hours, and then the pH of the solution is adjusted to 1. The method further comprises adjusting the pH to 0 to 3.0 and stirring for 1 to 24 hours.

[0039] Adjusting the pH to 7.0-8.0 neutralizes the solution and releases the depsy-peptide esters. The threonine and threonine impurities are converted to the desired compound.

[0040] Subsequently, when the pH is adjusted to 1.0–3.0, the Trp residue is decarboxylated, forming Trp CO The di-salt is converted to the desired product.

[0041] In one embodiment of the method of the present invention, the purification of the compound comprises the step of: The solution is subjected to chromatographic purification.

[0042] Preferably, the chromatographic purification is HPLC or reverse phase HPLC.

[0043] More preferably, the purification is carried out using (i) an aqueous solution of sodium hydroxide or sodium bicarbonate. A chromatography eluent is added to a solution containing the compound to form a sodium salt in the solution. (ii) precipitating the sodium salt of the compound from the solution; iii) filtering, washing and drying the precipitated sodium salt of the compound. Included.

[0044] The sodium salt provides improved solubility of the compound compared to the zwitterion or acetate form. Furthermore, precipitation of the sodium salt of the compound replaces expensive freeze-drying procedures.

[0045] In a further aspect of the invention, a compound of the formula: [ka] wherein PG1 is a base-stable side chain protecting group; PG2 is an ivDde, Dde, or Alloc side chain protecting group (SEQ ID NO: 17) a process for preparing a compound of formula (I), The method comprises: (i) the following formula: [ka] wherein PG1 is a base-stable side chain protecting group; PG2 is an ivDde, Dde, or Alloc side chain protecting group (SEQ ID NO: 9) and performing solid phase synthesis of a compound of formula (I) (ii) reacting the compound of step (i) with the compound of the formula: PG1-His(PG1)-Aib-Gln(PG1)-Gly-Thr(PG1) -OH wherein PG1 is a base-stable side chain protecting group (SEQ ID NO: 13). and c. coupling the signal to the signal.

[0046] In a preferred embodiment of the method of the present invention, PG1 is Boc in the case of Trp and Lys. and O in the case of Asp and Glu. t Bu, and Ser, Thr, and Tyr teeth t Bu, Trt for Gln, and Boc(Dnp) for His. be.

[0047] In a further preferred embodiment of the method of the present invention, PG2 is ivDde.

[0048] In an alternative preferred embodiment of the method of the invention, PG2 is Dde.

[0049] In a further aspect of the invention, a compound of the formula: [ka] wherein PG1 is a base-stable side chain protecting group; PG2 is an ivDde, Dde, or Alloc side chain protecting group (SEQ ID NO: 17) a process for preparing a compound of formula (I), The method comprises: (i) the following formula: [ka] wherein PG1 is a base-stable side chain protecting group; PG2 is an ivDde, Dde, or Alloc side chain protecting group (SEQ ID NO: 11). and performing solid phase synthesis of a compound of formula (I) above. (ii) reacting the compound of step (i) with the compound of the formula: PG1-His(PG1)-Aib-Gln(PG1)-Gly-OH wherein PG1 is a base-stable side chain protecting group (SEQ ID NO: 15). and c. coupling the signal to the signal.

[0050] In a preferred embodiment of the method of the present invention, PG1 is Boc in the case of Trp and Lys. and O in the case of Asp and Glu. t Bu, and Ser, Thr, and Tyr teeth t Bu, Trt for Gln, and Boc(Dnp) for His. be.

[0051] In a further preferred embodiment of the method of the present invention, PG2 is ivDde.

[0052] In an alternative preferred embodiment of the method of the present invention, PG2 is Dde.

[0053] In a further aspect of the invention, a compound of the formula: [ka] [wherein the lysine at position 20 (Lys / K) is in contact with the epsilon-amino group of the lysine side chain ([ 2-(2-aminoethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO-(C H2) 18 modified by bonding with COH (SEQ ID NO: 1) 1. A process for preparing a sodium salt comprising: The method comprises: (i) Adding an aqueous solution of sodium hydroxide or sodium bicarbonate to a solution containing the compound to form a sodium salt of the compound in solution; (ii) precipitating the sodium salt of the compound from the solution; (iii) filtering, washing and drying the precipitated sodium salt of the compound; A method is provided, comprising:

[0054] In a further aspect of the present invention, there is provided a compound having the following formula (SEQ ID NO:3): [ka]

[0055] In a further aspect of the present invention, there is provided a compound having the following formula (SEQ ID NO:4): [ka]

[0056] In a further aspect of the present invention, there is provided a compound having the following formula (SEQ ID NO:10): [ka]

[0057] In a further aspect of the present invention, there is provided a compound having the following formula (SEQ ID NO:12): [ka]

[0058] In a further aspect of the present invention, there is provided a compound having the following formula (SEQ ID NO:13): PG1-His(PG1)-Aib-Gln(PG1)-Gly-Thr(PG1)- OH where PG1 is a base-stable side chain protecting group.

[0059] Preferably, PG1 is, in the case of Thr, t Bu, and Trt in the case of Gln. In the case of His, it is Boc(Dnp).

[0060] In a further aspect of the present invention, there is provided a compound having the following formula (SEQ ID NO:15): PG1-His(PG1)-Aib-Gln(PG1)-Gly-OH wherein PG1 is a base-stable side chain protecting group.

[0061] Preferably, PG1 is Trt in the case of Gln and Boc(D) in the case of His. np). DETAILED DESCRIPTION OF THE INVENTION

[0062] As used herein, the following abbreviations have the meanings set forth herein: "SPPS" stands for solid phase peptide synthesis, and "Fmoc" stands for fluorenylmethyl. "Boc" means tert-butyloxycarbonyl chloride; means "O t "Bu" means tert-butyl ester, t Bu" is te "Trt" means rt-butyl, "Trt" means triphenylmethyl or trityl, and "D "np" means 2,4-dinitrophenyl, and "ivDde" means 1-(4,4-dinitrophenyl). means (2,6-dioxocyclohex-1-ylidene)-3-methylbutyl, "Dde" is (1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene) )-3-ethyl), "Alloc" means allyloxycarbonyl, and "P "ip" means piperidine, and "DIC" means diisopropylcarbodiimide. "Oxyma" means ethyl cyanohydroxyiminoacetate, and "DCM" means "IPA" means dichloromethane, "MTBE" means isopropanol "TFA" means trifluoroacetic acid. "TIPS" stands for triisopropylsilane, and "DTT" stands for dithiothreitol. "UPLC" stands for ultra-high performance liquid chromatography, and "HATU" stands for is (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4, 5-b]pyridinium 3-oxide hexafluorophosphate, "HFIP" means hexafluoroisopropanol, and "CTC" means chlorotrityl "AEEA" is 17-amino-10-oxo-3,6,12,15-tetraoxa-9 -azaheptadecanoic acid, "TMSA" means trimethylsilylamide, "HOBt" stands for hydroxybenzotriazole, and "API" stands for active pharmaceutical ingredient. "PyBOP" means (benzotriazol-1-yloxy)tripyrrolidino Phosphonium hexafluorophosphate) and " t BuO-C 20 -γGlu( t Bu)-AEEA-AEEA-OH" is (3,6,12,15-tetraoxa-9, 18-Diazatricosane diacid, 22-[[20-(1,1-dimethylethoxy)-1,2 0-Dioxoeicosyl]amino]-10,19-dioxo, 2,3-(1,1-dimethyl "AEEA" means (8-amino-3,6-diethyl) ester, (22S) dioxaoctanoic acid).

[0063] The amino acid sequences of the present invention are represented by the standard single-letter or triple-letter sequences of the 20 naturally occurring amino acids. It contains the letter code. Furthermore, "Aib" is alpha aminoisobutyric acid.

[0064] The present invention generally relates to Gcg and GLP-1 duplexes, wherein the compounds are synthesized by SPPS. The present invention is directed to a method for preparing an agonist compound. There are several basic steps that are repeated each time an additional amino acid is added to the nucleotide chain. The "solid phase" is the substrate to which the first amino acid and then the growing peptide chain is attached. The chains are attached to the particles, making them particularly easy to clean and separate, e.g. The chains can be treated as if they were aggregates of solid particles (e.g., during a filtration step), and Therefore, in many cases the overall process is simpler than pure solution synthesis.

[0065] There are several suitable resins for constructing the peptide compounds presented herein. For example, Sieber and Rink amide resins are well known for preparing peptides. However, alternative resins may be selected to prepare the peptides described herein. For example, but not limited to, 2-CTC and related resins may be used. The target peptide can then be prepared, followed by a C-terminal amidation step.

[0066] The repeated steps of SPPS include deprotection, activation, and coupling: (i) Deprotection: Before each cycle begins, the last acid in the peptide chain is "protected" As used herein, the term "protected" means that the protecting group remains that is, the "amino" terminus is protected from undesired reactions. A variety of protecting groups are well known and alternatives are available. Any suitable protecting group may be suitable for a particular method. A "protecting group" is a group that protects the amino acid residue of the next amino acid to be added. (the "deprotection" step). (ii) Activation: A compound (an "activator") is added to the reaction to activate the deprotected peptide on the peptide chain. This produces an intermediate amino acid species that is more likely to couple to the selected acid. (iii) Coupling: An activated species attaches to an existing peptide chain.

[0067] One of the most commonly used and studied activation methods in peptide synthesis is the calcitonin (Cal) activation method. The carbodiimide reacts with the carboxylic acid of an amino acid derivative to form a Contains two slightly basic nitrogen atoms that form highly reactive O-acylisourea compounds The O-acylisourea formed then reacts immediately with the amine to form the peptide. Alternatively, O-acylisourea can be converted to other reactive species. However, some of these alternative reactions of O-acylisourea Some promote undesired pathways, leading to peptide bond formation. Conversion to a non-reactive N-acylurea prevents coupling. On the other hand, epimerization of activated chiral amino acids occurs via oxazolone formation. By using an excess of amino acid compared to the carbodiimide, a more specific However, this approach is , undesirably consuming additional amino acid equivalents.

[0068] 1-Hydroxybenzotriazole (HOB) was used as an additive during the activation of carbodiimides. The incorporation of HOB has significantly improved the carbodiimide activation method. t rapidly converts O-acylisourea to the highly reactive OBt ester, but with undesired Avoids the formation of N-acylisourea and oxazolones. HOBt is suitable for large-scale commercial production. Instead of HOBt, ethyl 2-cyano-2- (hydroxyimino)acetate (Oxyma, OxymaPure, ECHA) or 1 Other additives such as 2,5-hydroxy-2,5-pyrrolidinedione (NHS) can be used. can.

[0069] For the method of the present invention, the preferred activation system is DIC / Oxyma in DMF. Preferably, the ratio of amino acid:oxygen:DIC is 2.0:2.0:2.2. All charges are based on the limiting reagent, an amide resin. Oxyma-based systems are Improve downstream aggregation and impurities observed in purification steps, especially chromatographic purification. Suitable solvents include DMF, NMP, and NBP. DMF is the preferred solvent system because it is significantly cheaper.

[0070] More generally, with respect to the methods of the present invention, the construction of SPPS is preferably carried out by automated peptide synthesis. This was achieved using standard Fmoc peptide chemistry techniques with sequential couplings on the synthesizer. The preferred resin is Sieberamide resin. DMF is the preferred solvent system. The resin is then swollen with DMF. The resin is deprotected preferably with 20% piperidine (Pi This is achieved using HCl / DMF (3 x 30 min). Subsequent Fmoc deprotection is preferably Preferably, 20% Pip / DMF (9 ml / g resin) 3 x 30 minute treatments are used. For more difficult couplings, it is advisable to use 4 x 30 min treatments. Afterwards, the resin is washed with 10 volumes of DMF for 6 x 2 min. For resin-bound peptides, use a DIC / Oxyma / DMF solution for 30 minutes at room temperature. Coupling of activated amino acids occurs for the time specified for each individual amino acid. After pulling, solvent washes are performed with 10 volumes of DMF for 6 x 2 min.

[0071] To isolate the final product, the resin-bound product is diluted 5x with preferably 10x the volume of DCM. Wash for 2 minutes to remove DMF. Wash the resin with preferably 10 volumes of IPA 2 x 2 minutes. Wash to remove DCM and add 10 volumes of methyl tert-butyl ether (MTBE). The resin-bound product is washed 5 x 2 minutes at 40°C and then vacuum dried at 40°C. Store in a cool, dry place (-20°C).

[0072] For analysis, peptides were diluted in the following ratios of TFA / H2O / TIPS / DTT: The cleavage from the resin is carried out using a preferred acid cocktail: (0.93v / 0.04v / 0 The resin is preferably swelled in DCM (4-5 mL, 3 x 30 Add the cleavage cocktail (4-5 mL) to the pre-swollen resin and suspend. The suspension is stirred at room temperature for 2 hours. The solution is filtered and the resin is then washed with water, preferably with a small amount of DCM. The resulting solution is then mixed with the cleavage solution, preferably in a volume of 7 to 10 times that of cold (0°C) Pour the suspension into methyl tert-butyl ether (MTBE). Allow to age for 10 minutes, then centrifuge the resulting precipitate and decant the clear solution. The distillate is suspended in preferably an equal volume of MTBE, and the resulting suspension is centrifuged again to extract decane. After decanting, the clear MTBE solution of precipitated peptide was dried under vacuum at 40°C overnight. To make.

[0073] The present invention relates to the compounds disclosed herein, or pharmaceutically acceptable salts thereof, particularly nanoparticles. The present invention is directed to novel compounds and methods useful for the synthesis of thorium salts. , as illustrated in the Examples below. The reagents and starting materials are readily available to one of ordinary skill in the art. It is understood that these embodiments are not intended to limit the scope of the invention in any way. do.

[0074] Example 1: Preparation of the compound of SEQ ID NO:1 Synthesis of Preparation 1 [ka] SEQ ID NO:3 Fmoc Sieber resin (0.6–0.8 mmol / g) was loaded into the reactor and swollen with DMF. The resin is then washed with DMF for 2 hours and stirred for 2 hours, after which the DMF is filtered off the resin. The Fmoc-protected resin was then washed twice with 20% Pip / DMF at 9 ml / g resin. Sampling to confirm Fmoc removal is performed using the final Pip After / DMF treatment, UV analysis was performed to confirm >99% Fmoc removal (I The goal for PC was less than 1% residual Fmoc). After the final 20% w / w Pip / DMF treatment Next, wash the resin bed multiple times with DMF (e.g., 6 x 2 min, 10 volumes at 9 ml / g resin). (DMF wash). The following conditions for coupling and deprotection of each amino acid were used: To build the peptide backbone: [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0075] Fmoc deprotection: The resin in the peptide reactor was loaded three or four times with 20% v / v Pip / DMF solution. Each treatment was stirred on the resin for 30 minutes, followed by filtration to complete the removal of the Fmoc protecting group. After the final 20% v / v PIP / DMF treatment, the resin bed is Wash a minimum of 6 times with MF volume loads of DMF.

[0076] Amino acid activation: A pre-prepared solution of 12% w / w Oxyma Pure / DMF was charged into the reactor. Then add the selected Fmoc amino acid. The mixture is stirred at 20±5°C until the slightly exothermic activation reaction is controlled. To ensure the Fmoc-AA / Oxyma Pure / DMF solution was The solution is cooled to 15±3°C and the resulting solution temperature is maintained within the specified range of 20±5°C. The solution is activated by adding DIC. The solution is transferred to the reactor containing the peptide on the resin compound. Stir the activated ester solution for 20-30 minutes before use.

[0077] Coupling: Once the activation step is complete, the activated ester solution is added to the deprotected peptide on the resin. The peptide coupling reaction is then carried out for 20± Stir for at least 4 hours at 5°C. After the required stirring time, coupling completion (IPC) is reached. Sample the resin slurry for IPC results as needed until a passing IPC result is obtained. Repeat sampling at specific intervals depending on the situation. If necessary, perform recoupling operation. Once coupling is complete, the solution contents of the peptide reactor are filtered and then the resin compound is added. The resulting peptide is washed several times with DMF in preparation for the next coupling.

[0078] Gly-Thr pseudoproline dipeptide for coupling at positions 4 and 5 , used in place of the individual Fmoc-L-Gly and Fmoc-L-Thr amino acids Fmoc-Gly-Thr[Ψ( Me,Me )Pro]-OH is the above coupling conditions to be coupled to Phe(6).

[0079] Alternative synthesis of preparation 1: In an alternative synthesis of Preparation 1, in the amino acid activation step, O Instead of xyma, HOBT in NMP is used. The activator is DIC. The ratio of amino acids, DIC, and HOBT was 3.0:3.3:3.0 (AA / DIC of 3.0). The solvent system is NMP. NMP is an alternative It is also a solvent system used in coupling and deprotection reactions in practical synthesis.

[0080] Synthesis of Preparation 2 [ka] SEQ ID NO:6

[0081] Lys(20)ivDde deprotection: Fully protected 34 amino acid on the resin Boc-His(1)-Gly(34) peptide backbone Selective deprotection of the 1-34 Lys(20)ivDde group of the amino acid is carried out. Using 8% w / w hydrazine hydrate in MF solution and stirring at ambient temperature for 4 hours The deprotection reaction was monitored by HPLC, and the amount of 1-3 remaining after deprotection was Aim for an IPC limit of less than 1% of the 4Lys(ivDde) ​​component. The strip (Preparation 2; SEQ ID NO:3) was washed repeatedly (8 times) with DMF to remove residual hydrazine. Completely remove the two pieces of the fully assembled preparation. Wash four times with IPA and then Dry below 40°C until the LOD is achieved. t BuO-C20- γGlu( t Before coupling with (Bu)-AEEA-AEEA-OH, Store refrigerated (-20°C).

[0082] Synthesis of Preparation 3 [ka] SEQ ID NO:8

[0083] Preparation 2 t BuO-C20 -γGlu( t Cupric amine (Bu)-AEEA-AEEA-OH Pulling: Side chain t BuO-C 20 -γGlu( t Bu)-AEEA-AEEA-OH (2.0 equivalents ) and PyBOP (3.0 equiv.) solids were charged to the reactor, followed by 1:1 DMF / D Charge CM and stir the mixture until dissolution occurs. 2,4,6 Collidine (3.0 equiv.) to initiate the formation of the active ester species. Stir the activated ester solution for 30 minutes. Stir the reaction slurry at 35°C for 18 hours. The slurry is sampled for completion of coupling (IPC) and, if necessary, passed at specific intervals as needed to achieve a result of IPC (less than 1% of preparation 2) Repeat the sampling.

[0084] Once coupling is complete, filter and discard the contents of the solution. Preparation 3 is washed multiple times with DMF and then IPA. Preparation 3 is washed with 1% or less LO Dry below 40°C until D is achieved. Preparation 3 is packaged prior to cleavage from the resin. Package and store refrigerated (-20°C).

[0085] Following the synthesis of Preparation 1, Preparation 2, and Preparation 3 as described above, 28 g of Sieber tree The fat (0.6 mmol / g) was processed into 85 g of resin compound (i.e., Preparation 3) (yield rate 73%).

[0086] Alternative synthesis of preparation 3: The peptide backbone is constructed according to the alternative synthesis of Preparation 1 as described above. The oc deprotection is carried out using 20% ​​by weight Pip / NMP. Post-deprotection washes include: DMF solvent is used. For coupling of N-terminal Boc-His-BOC-OH, DE A PT / DIEA activation system is used. The preformed activated ester is stirred in NMP. Add to the slurried resin.

[0087] Prepared by selective deprotection of Lys20 ivDde with hydrazine as described above After forming product 2, four individual side chain couplings were carried out sequentially to give the resin-bound construct. Each cycle utilized the PyBOP / DIEA coupling reagent pair. Three of the chain components were prepared following a typical deprotection, coupling, and DMF washing protocol. In the final cycle, the mono-t-butyl-protected carbon A few dozen fatty acid diacids were used as the final segment coupled to the γGlu side chain. This coupling was performed using a 75:25 w / w toluene:NMP solvent mixture. , ensuring that the fatty acid remains in solution throughout the coupling sequence.

[0088] By following the Alternative Synthesis of Preparation 1 and Alternative Synthesis of Preparation 3 as described above 1.4 kg of Sieber resin (0.6 mmol / g) was added to 4.6 kg of resin compound (i.e. Then, it is processed into preparation 3) (yield 79%).

[0089] Synthesis of Preparation 4 [ka] SEQ ID NO:1

[0090] Resin cleavage / deprotection: A cleavage cocktail consisting of TFA, TIPS, DTT, DCM, and water is prepared. Cool the flask to 15±5° C. The reagent charges are shown in the table below: [Table 2]

[0091] Preparation 3 is charged to the reactor, followed by the cleavage cocktail. The mixture is incubated at 23°C for 3 hours. The mixture is filtered and the spent resin is washed with DCM. Combine the M wash filtrate with the bulk deprotection solution and cool the contents to below -10°C. Cool to below -13℃ and feed to the low-temperature filtrate twice. The internal temperature of the liquid is maintained below 5°C. The initial MTBE charge is approximately 45% of the total MTBE charge. A soft precipitate formed near the end of the MTBE addition, but the solution The precipitate solution is then re-cooled to an internal temperature of -15±5°C. The MTBE addition was carried out at a rate about 5 to 10 times the initial MTBE feed rate, and the MTBE This constitutes approximately 55% of the total charge. Maintain the internal temperature of the precipitation slurry below 0°C during the addition. The resulting slurry was aged at -8±3°C for a minimum of 6 hours, then warmed to 0±3°C and further Aged for 2 hours before isolation.

[0092] The cold crude peptide slurry was filtered and the resulting wet cake was then washed with MTBE. The wet cake of Preparation 4 was then dried to an IPC target LOD value of less than 1%. do.

[0093] By following the synthesis of Preparation 4 above, Preparation 4 was prepared using 44% by weight and 65% HP. Produced in LC area percent purity. Content yield based on Sieber resin is 47%.

[0094] purification The zwitterionic form of Preparation 4 was purified by chromatography and subsequently lyophilized. Can.

[0095] Chromatography: 4.25 kg of Preparation 4 (potency 41%, active content 1.71 kg) (preparation of Preparation 1 above) Alternative Synthesis and Preparation 3) Prepared according to the alternative synthesis of 4 / 6 / 90 formic acid / acetone The compound was dissolved in a nitrile / water solution to form a 10 mg / mL solution, which was stirred for 4 hours. The tryptophan is then decarboxylated prior to chromatography. The results were analyzed by reversed-phase chromatography on a 15 cm column using 27 initial injections and 2 recycles. A total of 671 kg containing 1.43 kg of the compound of SEQ ID NO: 1 was obtained by processing The compound of SEQ ID NO: 1 was purified by 22 initial runs to produce a solution of 93% purity and 83% yield. Further reversed-phase chromatography on a 15 cm column using single injection and four recycles. to give 278 kg of solution containing 1.19 kg of the compound of SEQ ID NO:1. (98% purity, 93.6% yield). The enrichment chromatography was carried out in four initial injections, yielding 1.16 kg of the active peptide. This results in a total of 38.4 kg of solution with a purity of 98% and a yield of 93.6%.

[0096] Lyophilization: The chromatographically concentrated solution was heated to 35°C and then diluted with acetonitrile (50 volumes). Dilute the diluted peptide solution with SEQ ID NO: 1 at a feed rate of 100-150 g per minute. 5 g (95% purity) of the compound (zwitterionic form) is seeded, and then a precipitate is formed. Stir at 35 °C until 35 °C. While maintaining the temperature at 35 °C, add the second charge of acetonitrile ( The resulting slurry was aged at 35°C for 1 hour, cooled to 20°C, and The slurry is then aged for at least 1 hour. The slurry is then filtered and the isolated product is then added to the The dried product is then washed with acetonitrile and dried to an LOD of less than 1%. Wet the API powder in 29 volumes of high purity water to remove residual solvent. It was dissolved in a solution of 0.38% (w / w) ammonium acetate and then placed in high purity water. Add 1.33 volumes of 9.1% (w / w) ammonium hydroxide solution in equal portions to dissolve and and achieve a final solution pH in the range of pH 8.2 to pH 8.6.

[0097] Aqueous solutions of the compounds were filtered through 0.2 micron polyethersulfone filters, Meanwhile, freeze-drying trays are filled to contain approximately 0.9 kg of aqueous solution per tray. The product was lyophilized according to an automated program that included freezing the solution at -40°C. The primary freeze-drying is carried out at a temperature of -40°C and a vacuum of approximately 100 mTorr. After lyophilization, a stepwise ramp sequence was performed to increase the shelf temperature from -40°C to 0°C. Secondary drying was carried out at about 15 mTorr and 20°C to yield 412 g of compound of SEQ ID NO:1. The product was produced as a white solid in 98% purity and 95% yield.

[0098] Purification and synthesis of sodium salt Chromatography First-pass HPLC purification was performed with TF of 0.1 / 90 / 10. Mobile phase A: water / acetonitrile (v / v); TFA: 0.1 / 10 / 90 (v / v); Water / acetonitrile mobile phase B and Kromasil 100-10-C8 stationary phase were used. and implement it.

[0099] Second-pass HPLC purification was performed using a 90 / 10 50 mM weight fraction. ammonium carbonate, pH 7.6 / acetonitrile (v / v) mobile phase A (MP-A), and Transfer to 10 / 90 50 mM ammonium bicarbonate, pH 7.6 / acetonitrile (v / v) Mobile phase B (MP-B) was used on Kromasil 100-10-C8 as the stationary phase. This will be implemented.

[0100] Synthesis of sodium salt After chromatographic purification, 90% of the 50 mM ammonium acetate, pH 8.5 / 1 0% isopropyl alcohol (v / v) mobile phase A (MP-A), 10% 50 mM vinegar Mobile phase B (M ammonium chloride, pH 8.5 / 90% isopropyl alcohol (v / v) PB), and a second pass using Amberchrom CG300-M stationary phase. The composite solution is concentrated.

[0101] Based on the molar equivalents of acid functional groups present on the peptide molecule, a concentrated aqueous solution of sodium hydroxide was added. The peptide was loaded into a condensed solution and an equimolar amount of hydroxide (OH) was added to free the free carboxylic acid of the peptide. Neutralize the group, which is

number

[0102] Example 2: Preparation of the compound of SEQ ID NO:10 Synthesis of Preparation 5 [ka] SEQ ID NO:10 Fmoc Sieber resin (0.6–0.8 mmol / g) was loaded into the reactor and swollen with DMF. The resin is then washed with DMF for 2 hours and stirred for 2 hours, after which the DMF is filtered off the resin. The Fmoc-protected resin was then washed twice with 20% Pip / DMF at 9 ml / g resin. Sampling to confirm Fmoc removal is performed using the final Pip After DMF treatment, UV analysis confirms >99% Fmoc removal (IP The goal for C is less than 1% residual Fmoc). After the final 20% w / w Pip / DMF treatment, , wash the resin bed multiple times with DMF (e.g., 6 x 2 min, 9 ml / g resin, 10 volumes) DMF wash). The following conditions for coupling and deprotection of each amino acid were used: , constructing the peptide backbone: [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

[0103] Fmoc deprotection: The resin in the peptide reactor was loaded three or four times with 20% v / v Pip / DMF solution. Each treatment was stirred on the resin for 30 minutes, followed by filtration to complete the removal of the Fmoc protecting group. After the final 20% v / v PIP / DMF treatment, the resin bed is Wash a minimum of 6 times with MF volume loads of DMF.

[0104] Amino acid activation: A pre-prepared solution of 12% w / w Oxyma Pure / DMF was charged into the reactor. Then add the selected Fmoc amino acid. The mixture is stirred at 20±5°C until the slightly exothermic activation reaction is controlled. To ensure the Fmoc-AA / Oxyma Pure / DMF solution was The solution is cooled to 15±3°C and the resulting solution temperature is maintained within the specified range of 20±5°C. The solution is activated by adding DIC. The solution is transferred to the reactor containing the peptide on the resin compound. Stir the activated ester solution for 20-30 minutes before use.

[0105] Coupling: Once the activation step is complete, the activated ester solution is added to the deprotected peptide on the resin. The peptide coupling reaction is then carried out for 20± Stir for at least 4 hours at 5°C. After the required stirring time, coupling completion (IPC) is reached. Sample the resin slurry for IPC results as needed until a passing IPC result is obtained. Repeat sampling at specific intervals depending on the situation. If necessary, perform recoupling operation. Once coupling is complete, the solution contents of the peptide reactor are filtered and then the resin intermediate is removed. The resulting peptide is washed several times with DMF in preparation for the next coupling.

[0106] Example 3: Boc-His(Dnp)-Aib-Gln(Trt)-Gly-Thr ( t Preparation of (Bu)-OH pentamer (SEQ ID NO: 14) Synthesis of Preparation 6 Boc-His(Dnp)-Aib-Gln(Trt)-Gly-Thr( t Bu)-O H SEQ ID NO:14

[0107] Resin Loading: The amount of Fmoc-L-Thr(tBu)-OH on the CTC resin was added to reactors 1 to 3, respectively. Load one-third of the sieve (0.769 mmol / g, 100-200 mesh, 2.94 g) The resin was swelled in 3 x 15 ml DMF for 20 min each, and then 3 x Deprotect with 15 ml of 20% Pip / DMF for 30 min each before the first coupling. Then wash with 5 x 15 ml DMF for 1 min each.

[0108] Fmoc-Gly-OH coupling: The solution was added to 2-(9H-fluoro-2-methyl ... (9-phenyl-2-methoxycarbonylamino)acetic acid (2.01 g, 6.76 mmol) and From ethyl cyanoglyoxylate-2-oxime (960 mg, 6.688 mmol) Prepare N,N'-diisopropylcarbodiimide (1.17 mL, 7.47 mmol). l) is added to this pale yellow solution, and the orange-yellow solution is allowed to stand for 30 minutes with occasional shaking. One-third of the solution was added directly to each reactor by pipette, and the reactions were mixed for 12 hours. Drain. Wash the resin with 5 x 15 ml DMF for 1 min each, then with 4 x 15 ml 20 ml DMF. % Pip / DMF (v / v) for 30 min each, followed by deprotection with 5 × 15 ml D Wash with MF for 1 min each and proceed to the next coupling.

[0109] Fmoc-L-Gln(Trt)-OH coupling: The solution was dissolved in 40.5 ml of DMF in a 60 ml bottle. H-fluoren-9-ylmethoxycarbonylamino)-5-oxo-5-(tritylamino) (amino)pentanoic acid (4.12 g, 6.75 mmol) and ethyl cyanoglyoxylate Prepared from N,N'-diisopropyl-2-oxime (960 mg, 6.688 mmol). Propylcarbodiimide (1.17 mL, 7.47 mmol) was added to this pale yellow solution. Let the orange-yellow solution stand for 30 minutes, shaking occasionally. Pipette one-third of the solution into the The resin is added directly to each reactor and the reactants are mixed for 12 hours, then drained. Wash with 15 ml of DMF for 1 minute each, then 4 x 15 ml of 20% Pip / DMF ( v / v) for 30 min each, followed by 5 × 15 ml DMF for 1 min each Wash and proceed to next coupling.

[0110] Fmoc-Aib-OH coupling: The solution was added to 2-(9H-fluoro-2-methyl ... 2.20g, 6 .76 mmol) and ethyl cyanoglyoxylate-2-oxime (960 mg, 6. Prepared from N,N'-diisopropylcarbodiimide (1.17 mL, 7.47 mmol) was added to the pale yellow solution, and the orange-yellow solution was stirred occasionally. Add one-third of the solution directly to each reactor by pipette and let stand for 30 minutes. Mix for 18 hours and drain. Wash the resin with 5 x 15 ml DMF for 1 minute each. , 4 × 15 ml of 20% Pip / DMF (v / v) for 30 min each, then Wash with 5 x 15 ml DMF for 1 min each and proceed to the next coupling.

[0111] Boc-L-His(Dnp)-OH coupling: The solution was added to a 60 ml bottle containing Boc-His(d np)-OH (2.84 g, 6.74 mmol) and ethyl cyanoglyoxylate-2 Prepared from N,N'-diisopropyl ether (960 mg, 6.688 mmol). Add pyrcarbodiimide (1.17 mL, 7.47 mmol) to this bright yellow solution. The reaction mixture is mixed for 18 hours, and then one-third of the orange-yellow solution is immediately added to each reactor. Wash the resin with 5 x 15 ml DMF for 1 minute each, then 5 x 15 ml DCM for 1 minute each, then drain and dry for 4 hours.

[0112] Cleavage from the resin: The combined peptide on the resin was divided into two portions, and each portion was added to a 40 ml reaction vial. Add 30 ml of 30% hexafluoroisopropanol (HFIP) / DCM (v / v) to the flask. v) and mix for 2 hours on a rotary mixer. The resin is filtered off using a fritted filter. Wash twice with a total of 30 ml of DCM. Rotate the combined filtrate and wash Concentration by evaporation gave a dry yellow foam, which was then treated with methyl tert-butyl ether. Triturate twice with methyl ether (MTBE), rotating each time (to remove HFIP). Concentrate to dryness using an evaporator to obtain a bright yellow-orange powder solid. Trituration with cold 1:1 MTBE / heptane and sonication resulted in a yellow suspension The suspension is transferred to a centrifuge tube and centrifuged. If the solids do not settle well into a pellet, Because of the possibility of the solids being too hot, an additional 30 ml of cold MTBE / heptane was added and the solids were separated by a Buchner funnel. Filter through a funnel, wash with a small amount of cold 1:1 MTBE / heptane, and place in a vacuum oven at 35°C. After overnight drying, 2.255 g (91.4%) of a yellow solid was obtained with an UPL of 88.1%. It is obtained with C purity.

[0113] Example 4: Preparation of the compound of SEQ ID NO:12 Synthesis of Preparation 7 [ka] SEQ ID NO:12 Fmoc Sieber resin (0.6–0.8 mmol / g) was loaded into the reactor and swollen with DMF. The resin is then mixed with DMF and stirred for 2 hours, after which the DMF is filtered off from the resin. The Fmoc-protected resin is then washed twice with 20% Pip / DM at 9 ml / g resin. Deprotect using F treatment. Sampling to confirm Fmoc removal is performed using the final P After ip / DMF treatment, UV analysis confirms >99% Fmoc removal ( The goal for IPC was less than 1% residual Fmoc. Afterwards, the resin bed is washed multiple times with DMF (e.g., 6 x 2 min, 10 x 9 ml / g resin). (volume of DMF wash). The following conditions for coupling and deprotection of each amino acid were used: To build the peptide backbone: [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4]

[0114] Fmoc deprotection: The resin in the peptide reactor was loaded three or four times with 20% v / v Pip / DMF solution. Each treatment was stirred on the resin for 30 minutes, followed by filtration to complete the removal of the Fmoc protecting group. After the final 20% v / v PIP / DMF treatment, the resin bed is Wash a minimum of 6 times with MF volume loads of DMF.

[0115] Amino acid activation: A pre-prepared solution of 12% w / w Oxyma Pure / DMF was charged into the reactor. Then add the selected Fmoc amino acid. The mixture is stirred at 20±5°C until the slightly exothermic activation reaction is controlled. To ensure the Fmoc-AA / Oxyma Pure / DMF solution was The solution is cooled to 15±3°C and the resulting solution temperature is maintained within the specified range of 20±5°C. The solution is activated by adding DIC. The solution is transferred to the reactor containing the peptide on the resin compound. Stir the activated ester solution for 20-30 minutes before use.

[0116] Coupling: Once the activation step is complete, the activated ester solution is added to the deprotected peptide on the resin. The peptide coupling reaction is then carried out for 20± Stir for at least 4 hours at 5°C. After the required stirring time, coupling completion (IPC) is reached. Sample the resin slurry for IPC results as needed until a passing IPC result is obtained. Repeat sampling at specific intervals depending on the situation. If necessary, perform recoupling operation. Once coupling is complete, the solution contents of the peptide reactor are filtered and then the resin intermediate is removed. The resulting peptide is washed several times with DMF in preparation for the next coupling.

[0117] Example 5: Preparation of the compound of SEQ ID NO:16 Synthesis of Preparation 8 Boc-His(Dnp)-Aib-Gln(Trt)-Gly-OH SEQ ID NO:16

[0118] Resin Loading: Three separate fritted bottom reactors were filled with three portions of Fmoc-Gly-OH on CTC resin. Load each tube with 1 (100-200 mesh, 2.98 g, 2.25 mmol, 0. (756 mmol / g loading). Each resin was swelled in 3 x 15 ml DMF for 20 min each. and Fmoc deprotection with 3 x 15 ml of 20% piperidine / DMF (v / v) for 30 min. and washed with 5 x 15 ml DMF for 1 min each before the first coupling.

[0119] Fmoc-Gln(Trt)-OH coupling: The solution was dissolved in 40.5 ml of DMF in a 60 ml bottle. H-fluoren-9-ylmethoxycarbonylamino)-5-oxo-5-(tritylamino) (amino)pentanoic acid (4.12 g, 6.75 mmol) and ethyl cyanoglyoxylate Prepared from N,N'-di- Isopropylcarbodiimide (937.0 mg, 7.425 mmol, 100% by mass) Add this to the pale yellow solution and let the orange-yellow solution stand for 30 minutes with occasional shaking. One-third of the HCl solution was added directly to each reactor via pipette, and the reactions were mixed for 12 hours, then Wash the resin with 5 x 15 ml DMF for 1 minute each, then with 4 x 15 ml 2 Deprotection was performed with 0% Pip / DMF (v / v) for 30 min each, followed by 5 × 15 ml Wash with DMF for 1 minute each and proceed directly to the next coupling.

[0120] Fmoc-Aib-OH coupling: The solution was added to 2-(9H-fluoro-2-methyl ... 2.20g , 6.76 mmol) and ethyl cyanoglyoxylate-2-oxime (969.0 m g, 6.750 mmol) prepared from N,N'-diisopropylcarbodiimide ( To this pale yellow solution, 937.0 mg (7.425 mmol) was added, and the orange-yellow solution was slowly dissolved. Allow to stand for 30 minutes with shaking. Pipette one-third of the solution directly into each reactor. The reaction is mixed for 18 hours and then drained. The resin is washed with 5 x 15 ml of DMF. Wash with 4 x 15 ml of 20% Pip / DMF (v / v) for 1 minute each, then rinse with 3 x 15 ml of 20% Pip / DMF (v / v) for 3 minutes each. Deprotection was performed for 10 minutes, followed by 5 x 15 ml DMF washes for 1 minute each, followed by the next coupling. Proceed to the next step.

[0121] Boc-His(Dnp)-OH coupling: The solution was added to a 60 ml bottle containing Boc-His(D np)-OH (D, 2.84 g, 6.74 mmol) and ethyl cyanoglyoxylate Prepared from N,N'-di- Isopropylcarbodiimide (937.0 mg, 7.425 mmol) was added to this bright yellow Add one-third of the orange-yellow solution to each reactor immediately. Mix for 1 minute, then drain. Wash the resin with 5 x 15 ml DMF for 1 minute each. Wash with 5 x 15 ml DCM for 1 minute each, then drain and dry for 4 hours.

[0122] Cleavage of the peptide from the resin: The combined peptides on the resin from all three reactors were divided into two portions, and each portion was In a 40 ml reaction vial, add 30 ml of 30% hexafluoroisopropanol (H The resin was suspended in FIP / DCM (v / v) and mixed on a rotary mixer for 2 hours. Filter through a litre funnel and wash twice with a total of 30 ml of DCM. The purified solution was concentrated by rotary evaporation to a dry yellow foam and then Triturated twice with methyl tert-butyl ether (MTBE) (to remove residual HFIP). (2) Concentrate each mixture to dryness using a rotary evaporator to obtain a bright yellow-orange powder. The solid was triturated with 50 ml of 1:1 MTBE / heptane and sonicated, which resulted in A fine yellow suspension was produced. The suspension was transferred to a centrifuge tube and centrifuged. The supernatant was After decanting, the solid was washed twice with 30 ml of MTBE and partially washed with a stream of nitrogen. After drying, the solid was dried overnight in a vacuum oven at 35°C, yielding 1.89 g (8 7.8%) of a yellow solid is obtained with a UPLC purity of 97.66%.

[0123] Example 6: t BuO-C 20 -γGlu( t Preparation of (Bu)-AEEA-AEEA-OH Synthesis of Preparation 9 (3,6,12,15-tetraoxa-9,18-diazatricosane diacid, 22-[[2 0-(1,1-dimethylethoxy)-1,20-dioxoeicosyl]amino]-10, 19-Dioxo-, 2,3-(1,1-dimethylethyl) ester, (22S) [ka] The synthesis is carried out using an automated peptide synthesizer.

[0124] Preparation of solvents and reagents: Charge 20 L of DMF to the solvent reservoir.

[0125] Charge 4 L of 20% Pip / DMF solution into the piperidine reservoir.

[0126] Using HATU (67.53 g, 177.6 mmol, 100% by weight) and DMF , 444 mL of 0.4 M HATU solution is prepared and then loaded into an appropriate solvent bottle.

[0127] N,N-Diisopropylethylamine (77.55 mL, 445 mmol, 100 mass % %) and DMF to prepare 444 mL of a 1.0 M DIEA solution, followed by the appropriate Load into a suitable solvent bottle.

[0128] Charge 4 L of CH2Cl2 into the DCM solvent bottle. Add 1 L of CH2Cl2 to the second DCM solvent bottle. Load M solvent bottle.

[0129] Preparation of amino acid solutions: 0.400M t BuO-C 20 137 mL of the -OH solution was added to 20-tert-butoxy -20-oxo-icosanoic acid (21.843g, 54.80mmol, 100% by mass) and and a DMF / toluene mixture (1:1) and then charged into the addition bottle.

[0130] 0.400M FmocNH-Glu-O t 137 mL of Bu solution was added to (4R)-5-t ert-Butoxy-4-(9H-fluoren-9-ylmethoxycarbonylamino)-5 -oxopentanoic acid (23.316 g, 54.80 mmol, 100% by weight) and DMF and loaded into the addition bottle.

[0131] 137 mL of 0.400 M FmocNH-AEEA-OH solution was added to 2-[2-[2-( 9H-Fluoren-9-ylmethoxycarbonylamino)ethoxy]ethoxy]acetic acid (2 Prepared from 1.121 g, 54.80 mmol, 100% by weight, and DMF in an addition bottle. Load into.

[0132] Coupling conditions were as follows: 0.133M, 2.0 equivalents of HATU, 5 Desorption / desorption with 0.0 equiv. DIEA, ambient temperature, 3 h, 20% piperidine / DMF 3 x 15 min protection.

[0133] Resin Loading: In this synthesis, 2-CTC resin (0.99 mmol / g) was used, and FmocNH-A Load with EEA] Add 1.01 g to each of 24 parallel reactions.

[0134] Symphony X automated program (per 1.0 mmol scale reaction): (i) Swelling: 3 x 15 mL DMF over 10 minutes (ii) Cycle: -3 x 15ml 20% Pip / DMF for 15 minutes each - 5 x 15 mL DMF washes for 30 seconds each -5mL of amino acids -5mL of DIEA -5mL of HATU -Stirring for 3 hours - 5 x 15 mL DMF washes for 30 seconds each (iii) Drying: - 5 x 15 mL methylene chloride for 30 seconds each -Drain for 2 hours

[0135] Cleavage Protocol: The combined lots were then soaked in 30% HFIP / CH2Cl2 (240 mL) for 1.5 h. The resin is cleaved by stirring. The resin is filtered and further CH2Cl2 (2 x 5 0 mL) and the solvent is removed from the filtrate under vacuum. The resulting oil is reconstituted in acetonitrile. The procedure was repeated to obtain 30.47 g (14% of the theoretical yield). 6%) of a viscous yellow oil, which was 52.3 area % of the desired product by UPLC analysis. It contained the product.

[0136] Chromatography: The crude product (30.47 g, 52.3 area % purity) was purified by flash chromatography ( 500 grams of silica gel, 85% dichloromethane / 10% methanol / 5% acetic acid Purify by elution with 1000 ribonucleotides (collecting 38 x 100 ml fractions). The desired product is obtained in fractions 17-19. It elutes at 34 and there are several mixed fractions before and after the clean product which is discarded. Fraction 17 34 was concentrated under reduced pressure to a pale yellow viscous liquid, and the remaining acetic acid was then added to hexane under reduced pressure. The acetone was removed by azeotropic distillation twice with acetone to give 17.94 g of purified product. Obtained as a pale yellow viscous oil with a purity of 0.6 HPLC area %.

[0137] Crystallization: The chromatographic concentrate (17.94 g) was placed in a 250 ml Erlenmeyer flask. Dissolve in 120 ml of acetonitrile in a container and heat the mixture until a pale yellow solution is formed. Stir at ambient temperature for about 10 minutes. Cool the solution to -20 to -25°C for about 4 hours. A solid will settle and will be particularly thick on the inside surface of the flask. Use a spatula to break up the solid. This will result in a well-dispersed suspension. Keep the solids at -20 to -25°C. The fritted glass filter and acetonitrile for cleaning were kept in a freezer at -20 to -25°C. The suspension is quickly filtered and washed with approximately 50 ml of cold acetonitrile. Quickly scrape the solids from the filter and transfer them to a glass bottle. A colorless oil forms which solidifies when cooled to -20°C. The total yield of Preparation 9 is 13.4 g (yield The yield was 74.7%) and the UPLC purity was 91.65 area %.

[0138] array 1) SEQ ID NO: 1 [ka] [Wherein, lysine (Lys / K) at position 20 is bonded to the epsilon-amino group of the lysine side chain ([2 -(2-aminoethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO—(CH 2) 18 modified by bonding with CO2H]

[0139] 2) SEQ ID NO: 2 [ka] wherein PG1 is a base-stable side chain protecting group; Thr at position 5 is optionally protected by PG1, PG2 is an ivDde, Dde, or Alloc side chain protecting group.

[0140] 3) SEQ ID NO: 3 [ka]

[0141] 4) SEQ ID NO: 4 [ka]

[0142] 5) SEQ ID NO: 5 [ka] wherein PG1 is a base-stable side chain protecting group; Thr at position 5 is optionally protected in PG1]

[0143] 6) SEQ ID NO: 6 [ka]

[0144] 7) SEQ ID NO: 7 [ka] wherein PG1 is a base-stable side chain protecting group; Thr at position 5 is optionally protected in PG1]

[0145] 8) SEQ ID NO: 8 [ka]

[0146] 9) SEQ ID NO: 9 [ka] wherein PG1 is a base-stable side chain protecting group; PG2 is an ivDde, Dde, or Alloc side chain protecting group.

[0147] 10) SEQ ID NO: 10 [ka]

[0148] 11) SEQ ID NO: 11 [ka] wherein PG1 is a base-stable side chain protecting group; PG2 is an ivDde, Dde, or Alloc side chain protecting group.

[0149] 12) SEQ ID NO: 12 [ka]

[0150] 13) SEQ ID NO: 13 PG1-His(PG1)-Aib-Gln(PG1)-Gly-Thr(PG1)-O H wherein PG1 is a base-stable side chain protecting group.

[0151] 14) SEQ ID NO: 14 Boc-His(Dnp)-Aib-Gln(Trt)-Gly-Thr( t Bu)-O H

[0152] 15) SEQ ID NO: 15 PG1-His(PG1)-Aib-Gln(PG1)-Gly-OH wherein PG1 is a base-stable side chain protecting group.

[0153] 16) SEQ ID NO: 16 Boc-His(Dnp)-Aib-Gln(Trt)-Gly-OH

[0154] 17) SEQ ID NO: 17 [ka] wherein PG1 is a base-stable side chain protecting group; PG2 is an ivDde, Dde, or Alloc side chain protecting group.

[0155] 18) SEQ ID NO: 18 [ka]

[0156] 19) SEQ ID NO: 19 [ka]

Claims

1. The following formula: 【Chemistry 1】 [wherein Lys at position 20 is a hydroxyl group of the epsilon-amino group of the Lys side chain and ([2-(2-amino (aminoethoxy)-ethoxy]-acetyl) ２ -(γ-Glu)-CO-(CH ２ ) １８ C O ２ H (SEQ ID NO: 1) The method of The method comprises: (i) a compound of the formula: 【Chemistry 2】 wherein PG1 is a base-stable side chain protecting group; Thr at position 5 may be protected by PG1; PG2 is an ivDde, Dde, or Alloc side chain protecting group (SEQ ID NO: 2) and performing solid phase synthesis of a compound of formula (I): (ii) Selectively deprotecting the Lys to obtain Lys-NH ２ (SEQ ID NO: 5) ｔ BuO-C ２０ -γGlu ( ｔ Bu)-AEEA-AEEA-OH By this, the compound is selectively acyl-coated at the Lys at position 20 (SEQ ID NO: 7). and (iii) cleaving the acylated compound from the solid support and removing any remaining side chain protecting groups; and (iv) purifying said compound.

2. PG1, (a) Boc for Trp and Lys; (b) O in the case of Asp and Glu ｔ Bu, (c) for Ser, Thr, and Tyr: ｔ Bu, (d) Trt in the case of Gln; The method according to claim 1, wherein (e) in the case of His, it is di-Boc.

3. 3. The method of claim 1 or 2, wherein PG2 is ivDde.

4. 3. The method of claim 1 or 2, wherein PG2 is Dde.

5. The Lys at position 20 is selectively deprotected by reaction with a solution containing hydrazine hydrate. The method according to claim 3 or 4,

6. The solution may be 1% to 15% w / w hydrazine in DMF, NMP, NBP, or DMSO. The method of claim 5, comprising adding gin hydrate.

7. 7. The method of claim 5, wherein the solution comprises 8% w / w hydrazine hydrate in DMF. How to do it.

8. 3. The method of claim 1 or 2, wherein PG2 is Alloc.

9. The Lys at position 20 can be substituted with a scavenger, preferably H ３ N.BH3, Me ２ NH・BH3, also PhSiH ３ In the presence of Pd(PPh ３ ) ４ The compound is selectively deprotected by reaction with The method according to claim 5.

10. PG1, (a) Boc for Trp and Lys; (b) O in the case of Asp and Glu ｔ Bu, (c) for Ser, Thr, and Tyr: ｔ Bu, (d) Trt in the case of Gln; (e) di-Boc for His; PG2 is ivDde, The solid phase synthesis of the compound (SEQ ID NO: 3) in step (i) comprises an Fmoc amide bond. The following sequential couplings are carried out on a solid support: Fmoc deprotection of the amide resin and ： (01) Fmoc-L-Gly-OH, (02)Fmoc-L-Ser( ｔ Bu)-OH (03)Fmoc-L-Ser( ｔ Bu)-OH (04) Fmoc-L-Pro-OH, (05) Fmoc-L-Gly-OH, (06) Fmoc-L-Gly-OH, (07)Fmoc-L-Glu(O ｔ Bu)-OH、 (08) Fmoc-L-Leu-OH, (09) Fmoc-L-Leu-OH, (10) Fmoc-L-Trp(Boc)-OH, (11)Fmoc-L-Glu(O ｔ Bu)-OH、 (12) Fmoc-L-Val-OH, (13) Fmoc-L-Phe-OH, (14)Fmoc-L-Glu(O ｔ Bu)-OH、 (15) Fmoc-Lys(ivDde)-OH, (16) Fmoc-L-Ala-OH, (17) Fmoc-L-Lys(Boc)-OH, (18) Fmoc-L-Lys(Boc)-OH, (19)Fmoc-L-Glu(O ｔ Bu)-OH (20)Fmoc-L-Asp(O ｔ Bu)-OH (21) Fmoc-L-Leu-OH, (())__________________________ ｔ ｢)!H、 (23) Fmoc-L-Lys(Boc)-OH, (24)Fmoc-L-Ser( ｔ Bu)-OH (())_______________________ ｔ ｢)!H、 (26)Fmoc-L-Asp(O ｔ Bu)-OH、 (27)Fmoc-L-Ser( ｔ Bu)-OH (28)Fmoc-L-Thr( ｔ Bu)-OH、 (29) Fmoc-L-Phe-OH, (30)Fmoc-Gly-Thr(ψ Ｍｅ，Ｍｅ Pro)-OH、 (31) Fmoc-L-Gln(Trt)-OH, (32) Fmoc-Aib-OH, and (33) Any one of claims 1 to 7, comprising Boc-L-His(Boc)-OH. The method described below.

11. PG1, (a) Boc for Trp and Lys; (b) O in the case of Asp and Glu ｔ Bu, (c) for Ser, Thr, and Tyr: ｔ Bu, (d) Trt in the case of Gln; (e) Boc(Dnp) for His; PG2 is ivDde, The solid phase synthesis of the compound (SEQ ID NO: 4) in step (i) comprises an Fmoc amide bond. The following sequential couplings are carried out on a solid support: Fmoc deprotection of the amide resin and ： (01) Fmoc-L-Gly-OH, (02)Fmoc-L-Ser( ｔ Bu)-OH (03)Fmoc-L-Ser( ｔ Bu)-OH (04) Fmoc-L-Pro-OH, (05) Fmoc-L-Gly-OH, (06) Fmoc-L-Gly-OH, (07)Fmoc-L-Glu(O ｔ Bu)-OH、 (08) Fmoc-L-Leu-OH, (09) Fmoc-L-Leu-OH, (10) Fmoc-L-Trp(Boc)-OH, (11)Fmoc-L-Glu(O ｔ Bu)-OH、 (12) Fmoc-L-Val-OH, (13) Fmoc-L-Phe-OH, (14)Fmoc-L-Glu(O ｔ Bu)-OH、 (15) Fmoc-Lys(ivDde)-OH, (16) Fmoc-L-Ala-OH, (17) Fmoc-L-Lys(Boc)-OH, (18) Fmoc-L-Lys(Boc)-OH, (19)Fmoc-L-Glu(O ｔ Bu)-OH (20)Fmoc-L-Asp(O ｔ Bu)-OH (21) Fmoc-L-Leu-OH, (())__________________________ ｔ ｢)!H、 (23) Fmoc-L-Lys(Boc)-OH, (24)Fmoc-L-Ser( ｔ Bu)-OH (())_______________________ ｔ ｢)!H、 (26)Fmoc-L-Asp(O ｔ Bu)-OH、 (27)Fmoc-L-Ser( ｔ Bu)-OH (28)Fmoc-L-Thr( ｔ Bu)-OH、 (29) Fmoc-L-Phe-OH, (30) Boc-His(Dnp)-Aib-Gln(Trt)-Gly-Thr( ｔ The method of any one of claims 1 to 7, comprising a hydroxyl group selected from the group consisting of hydroxyl groups, ...

12. PG1, (a) Boc for Trp and Lys; (b) O in the case of Asp and Glu ｔ Bu, (c) for Ser, Thr, and Tyr: ｔ Bu, (d) Trt in the case of Gln; (e) Boc(Dnp) for His; PG2 is ivDde, The solid phase synthesis of the compound (SEQ ID NO: 4) in step (i) comprises an Fmoc amide bond. The following sequential couplings are carried out on a solid support: Fmoc deprotection of the amide resin and ： (01) Fmoc-L-Gly-OH, (02)Fmoc-L-Ser( ｔ Bu)-OH (03)Fmoc-L-Ser( ｔ Bu)-OH (04) Fmoc-L-Pro-OH, (05) Fmoc-L-Gly-OH, (06) Fmoc-L-Gly-OH, (07)Fmoc-L-Glu(O ｔ Bu)-OH、 (08) Fmoc-L-Leu-OH, (09) Fmoc-L-Leu-OH, (10) Fmoc-L-Trp(Boc)-OH, (11)Fmoc-L-Glu(O ｔ Bu)-OH、 (12) Fmoc-L-Val-OH, (13) Fmoc-L-Phe-OH, (14)Fmoc-L-Glu(O ｔ Bu)-OH、 (15) Fmoc-Lys(ivDde)-OH, (16) Fmoc-L-Ala-OH, (17) Fmoc-L-Lys(Boc)-OH, (18) Fmoc-L-Lys(Boc)-OH, (19)Fmoc-L-Glu(O ｔ Bu)-OH (20)Fmoc-L-Asp(O ｔ Bu)-OH (21) Fmoc-L-Leu-OH, (())__________________________ ｔ ｢)!H、 (23) Fmoc-L-Lys(Boc)-OH, (24)Fmoc-L-Ser( ｔ Bu)-OH (())_______________________ ｔ ｢)!H、 (26)Fmoc-L-Asp(O ｔ Bu)-OH、 (27)Fmoc-L-Ser( ｔ Bu)-OH (28)Fmoc-L-Thr( ｔ Bu)-OH、 (29) Fmoc-L-Phe-OH, (30) Fmoc-L-Thr( ｔ Bu)-OH, and (31) Containing Boc-His(Dnp)-Aib-Gln(Trt)-Gly-OH The method according to any one of claims 1 to 7.

13. the resin solid support is an Fmoc amide resin solid support, and the solid phase synthesis is The method of any one of claims 10 to 12, comprising Fmoc deprotection of

14. 14. The method of claim 13, wherein the Fmoc amide resin solid support is Sieber resin.

15. Step (iii) adjusts the pH of the solution containing the cleaved and deprotected compound to between 7.0 and 8. 0 and stirring for 1 to 24 hours, followed by adjusting the pH of the solution to 1.0 to 3.0; 15. The method of any one of claims 1 to 14, further comprising stirring for 1 to 24 hours.

16. Purification of the compound may involve chromatographing the compound produced by step (iii).

16. The method of any one of claims 1 to 15, comprising subjecting the roughy to refining.

17. 17. The method of claim 16, wherein the chromatographic purification is HPLC or reverse phase HPLC. method.

18. The purification step comprises (i) adding a solution containing aqueous sodium hydroxide or aqueous sodium bicarbonate to the solution. A chromatography eluent is added to form the sodium salt of the compound in solution. (ii) precipitating the sodium salt of said compound from the solution; i) filtering, washing and drying the precipitated sodium salt of said compound; 18. The method of claim 16 or 17, comprising:

19. The following formula: 【Transformation 3】 wherein PG1 is a base-stable side chain protecting group; PG2 is an ivDde, Dde, or Alloc side chain protecting group (SEQ ID NO: 17) a process for preparing a compound of formula (I), The method comprises: (i) a compound of the formula: 【Chemistry 4】 wherein PG1 is a base-stable side chain protecting group; PG2 is an ivDde, Dde, or Alloc side chain protecting group (SEQ ID NO: 9) and performing solid phase synthesis of a compound of formula (I): (ii) combining the compound of step (i) with the following formula: PG1-His(PG1)-Aib-Gln(PG1)-Gly-Thr(PG1) -OH wherein PG1 is a base-stable side chain protecting group (SEQ ID NO: 13). and a coupling step.

20. PG1, (a) Boc for Trp and Lys; (b) O in the case of Asp and Glu ｔ Bu, (c) for Ser, Thr, and Tyr: ｔ Bu, (d) Trt in the case of Gln; 20. The method of claim 19, wherein (e) His is Boc(Dnp).

21. 21. The method of claim 19 or 20, wherein PG2 is ivDde.

22. 21. The method of claim 19 or 20, wherein PG2 is Dde.

23. The following formula: 【Transformation 5】 wherein PG1 is a base-stable side chain protecting group; PG2 is an ivDde, Dde, or Alloc side chain protecting group (SEQ ID NO: 17) a process for preparing a compound of formula (I), The method comprises: (i) a compound of the formula: 【Transformation 6】 wherein PG1 is a base-stable side chain protecting group; PG2 is ivDde, Dde, or Alloc side chain protecting group (SEQ ID NO: 11). and performing solid phase synthesis of a compound of formula (I) (ii) combining the compound of step (i) with the following formula: PG1-His(PG1)-Aib-Gln(PG1)-Gly-OH wherein PG1 is a base-stable side chain protecting group (SEQ ID NO: 15). and a coupling step.

24. P, (a) Boc for Trp and Lys; (b) O in the case of Asp and Glu ｔ Bu, (c) for Ser, Thr, and Tyr: ｔ Bu, (d) Trt in the case of Gln; 24. The method of claim 23, wherein (e) His is Boc(Dnp).

25. 25. The method of claim 23 or 24, wherein PG2 is ivDde.

26. 25. The method of claim 23 or 24, wherein PG2 is Dde.

27. The following formula: 【Transformation 7】 [wherein the lysine at position 20 (Lys / K) is in contact with the epsilon-amino group of the lysine side chain ( [2-(2-aminoethoxy)-ethoxy]-acetyl ２ -(γ-Glu)-CO-( CH ２ ) １８ CO ２ H (SEQ ID NO: 1) 1. A process for preparing the sodium salt of The method comprises: (i) adding an aqueous solution of sodium hydroxide or sodium bicarbonate to a solution containing the compound of SEQ ID NO: 1; adding an aqueous sodium solution to form the sodium salt of the compound in solution; (ii) precipitating the sodium salt of said compound from the solution; (iii) filtering, washing and drying the precipitated sodium salt of the compound of SEQ ID NO:1; and causing the

28. A compound having the following formula (SEQ ID NO:3): 【Transformation 8】

29. A compound having the following formula (SEQ ID NO:4): 【Chemistry 9】

30. A compound having the following formula (SEQ ID NO:10): 【Chemistry 10】

31. A compound having the following formula (SEQ ID NO:12): 【Chemistry 11】

32. A compound having the following formula (SEQ ID NO:13): PG1-His(PG1)-Aib-Gln(PG1)-Gly-Thr(PG1) -OH where PG1 is a base-stable side chain protecting group.

33. When PG1 is Thr, ｔ Bu, Gln is Trt, His is 33. The compound of claim 32, wherein Boc(Dnp) is

34. A compound having the following formula (SEQ ID NO:15): PG1-His(PG1)-Aib-Gln(PG1)-Gly-OH where PG1 is a base-stable side chain protecting group.

35. PG1 is Trt when Gln, and Boc(Dnp) when His 35. The compound of claim 34.

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