Synthetic Process for the Production of Modified GCC Receptor Agonists

Abstract

The present invention relates to a method for producing a synthetic peptide of SEQ ID NO: 1 or a pharma- ceutically acceptable salt thereof. The method described herein begins by (i) chemically synthesizing a linear peptide C-terminally attached to a solid support using a plurality of amino acids and at least one polyamino acid synthon, the linear peptide having protecting groups at one or more amino acids and / or the polyamino acid synthon. In some embodiments, at least one amine group of the polyamino acid synthon has a different protecting group than the N-terminus of the linear peptide.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 282,842, filed November 24, 2021, and U.S. Provisional Application No. 63 / 323,552, filed March 25, 2022, the contents of which are incorporated by reference in their entireties herein.

[0002] Technical Field The present invention relates to a method for producing a synthetic peptide of SEQ ID NO:1 or a pharma- ceutically acceptable salt thereof.

[0003] Sequence Listing This application incorporates by reference in its entirety the Sequence Listing entitled "223355-519432.xml" (7.73 kilobytes), which was created on Nov. 21, 2022 at 9:49 AM and was submitted electronically herewith. [Background technology]

[0004] 2. Background of the Invention Interstitial cystitis / bladder pain syndrome (IC / BPS) is a chronic condition characterized by bladder pain, usually accompanied by urgency, increased frequency and / or nocturia. IC / BPS is often misdiagnosed as a urinary tract infection, and antibiotics are generally ineffective. It is estimated that 3-7% of women and 3-4% of men meet the definition of IC / BPS. There may be several contributing factors to the cause of IC / BPS, and it is unclear whether IC / BPS is a primary disorder or a secondary consequence of another disorder [Hanno et al., 2015, 193;1545-1553]. There is no diagnostic test for IC / BPS, and diagnosis is generally based on urinary symptoms of urgency and frequency with pain associated with the bladder. Diagnosis is generally withheld until other conditions that may be causing these symptoms have been ruled out.

[0005] There are few approved treatments available for IC / BPS. Patients often begin treatment with non-pharmacological measures (general relaxation, stress management, behavioral modification, and physical therapy techniques). Due to the few effective treatments available for IC / BPS, many patients utilize off-label treatments, including intravesical instillations (i.e., a mixture of medications delivered directly to the bladder via a catheter) to relieve symptoms. More effective and well-tolerated treatments for IC / BPS are needed.

[0006] A 13 amino acid guanylate cyclase C (GC-C) agonist synthetic peptide is being developed for the treatment of bladder pain associated with IC / BPS, and potentially other visceral pain conditions in the abdominal region. To further develop this peptide, an efficient synthesis and purification process is needed. Summary of the Invention [Means for solving the problem]

[0007] Summary of the Invention The present invention relates to a method for producing a synthetic peptide, or a pharma- ceutically acceptable salt thereof, comprising the steps of: (i) chemically synthesizing a linear peptide C-terminally bound to a solid support using a plurality of amino acids and at least one polyamino acid synthon, the linear peptide having protecting groups on one or more amino acids and / or the polyamino acid synthon, and at least one amine group of the polyamino acid synthon having a different protecting group than the N-terminus of the linear peptide; (ii) cleaving the linear peptide from the solid support to produce a protected peptide; (iii) coupling an amino acid to the C-terminus of the protected peptide; and (iv) isolating one amine protecting group and one carboxylic acid protecting group of the protected peptide. A method comprising the steps of removing protecting groups to form a partially unprotected peptide having an unprotected amine and an unprotected carboxylic acid group, (v) coupling the unprotected amine and the unprotected carboxylic acid group to form a cyclized peptide, (vi) globally deprotecting the cyclized peptide to obtain a globally deprotected peptide, (vii) folding the globally deprotected peptide to form one or more additional crosslinks to obtain a synthetic peptide, (viii) optionally modifying the N-terminus of the synthetic peptide with one or more chemical moieties, and (ix) purifying the synthetic peptide. The synthetic peptide produced by the methods described herein has the amino acid sequence: Cys1Cth2Glu3Leu4Cys5Cys6Asn7Val8Ala9Cys 10 Tyr 11 Gly 12 Cys 13 (SEQ ID NO: 1). The synthetic peptides include the following synthetic peptides: Cys1 and Cys6, Cth2 and Cys 10 , and Cys5 and Cys 13 It contains covalent bonds between amino acid residues.

[0008] In some embodiments, the method includes the optional step of (viii) modifying the N-terminus of the synthetic peptide with one or more chemical moieties.

[0009] Also, the following structural formula: [ka] (In the formula, P 1 and P 2 is hydrogen or an amine protecting group, with the proviso that P 1 and P 2 If both are amine protecting groups, then they are not the same amine protecting group, and P 3 is hydrogen or a carboxylic acid protecting group, P 4 is hydrogen or a thiol protecting group), or a pharma- ceutically acceptable salt thereof. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 shows an exemplary flow diagram for the production of the synthetic peptide of SEQ ID NO:1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Detailed Description of the Invention Described herein are methods for producing a synthetic peptide, or a pharma- ceutically acceptable salt thereof. The methods described herein include: (i) chemically synthesizing a linear peptide C-terminally attached to a solid support using a plurality of amino acids and at least one polyamino acid synthon, the linear peptide having protecting groups on one or more of the amino acids and / or polyamino acid synthons; chemically synthesizing a polyamino acid synthon in which at least one amine group has a different protecting group than the N-terminus of the linear peptide; (ii) cleaving the linear peptide from the solid support to produce a protected peptide; (iii) coupling an amino acid to the C-terminus of the protected peptide, the amino acid having an unprotected amine group, a protected carboxylic acid group, and an optionally protected amino acid side chain; (iv) removing one amine protecting group and one carboxylic acid protecting group of the protected peptide to form a partially unprotected peptide having an unprotected amine and an unprotected carboxylic acid group; (v) coupling an unprotected amine with an unprotected carboxylic acid group to form a cyclized peptide; (vi) globally deprotecting the cyclized peptide to obtain a globally deprotected peptide; (vii) folding the globally deprotected peptide to form one or more additional crosslinks to obtain a synthetic peptide; (viii) optionally modifying the N-terminus of the synthetic peptide with one or more chemical moieties; (ix) purifying the synthetic peptide, The synthetic peptide has the amino acid sequence: Cys1Cth2Glu3Leu4Cys5Cys6Asn7Val8Ala9Cys 10 Tyr 11 Gly 12 Cys 13 (SEQ ID NO: 1), The synthetic peptide comprises the following: a) Cys1 and Cys6, b) Cth2 and Cys 10 and c) Cys5 and Cys 13 It contains covalent bonds between amino acid residues.

[0012] definition As used herein, "Cth" represents cystathionine, which has two α-aminocarboxyl groups, designated "1" and "2" in Scheme 1, that are capable of forming a peptide bond. [ka]

[0013] However, to facilitate the use of the three-letter amino acid code in writing peptide sequences, when a cyclic peptide sequence is created by forming a peptide bond with each of the α-amino carboxyl groups (designated "1" and "2") at discrete positions in the peptide sequence to create a cyclic thioether bridge, the peptide bond formed by the α-amino carboxyl group at position 1 is designated "Cth" and the peptide bond formed by the α-amino carboxyl group at position 2 is designated "Cys." For further details, see the section entitled "Synthetic Peptides."

[0014] As used herein, "Hcy" refers to homocysteine ​​as shown in Scheme 1. As can be seen from Scheme 1, cystathionine can be considered as a combination of homocysteine ​​and cysteine, whose side chains share a sulfur atom. Thus, an alternative way of designating a cyclic peptide sequence created by forming peptide bonds with each of the α-aminocarboxyl groups of cystathionine at discrete positions in the peptide sequence is to designate the peptide bond formed by the α-aminocarboxyl group at position 1 as "Hcy" and the peptide bond formed by the α-aminocarboxyl group at position 2 as "Cys."

[0015] As used herein, unless otherwise indicated, "pharmacologically acceptable" means biologically or pharmacologically compatible for in vivo use in animals or humans, and preferably means approved by a regulatory agency of a federal or state government or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals, and more specifically in humans.

[0016] As used herein, unless otherwise indicated, the terms "about" and "approximately" refer to within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within one standard deviation or more than one standard deviation, as per the convention in the art. Alternatively, with respect to a composition, "about" can mean plus or minus a range of up to 20%, preferably up to 10%. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, more preferably within 2-fold, of a value. Particular values ​​are described in the present application and claims, and unless otherwise indicated, the term "about" refers to within an acceptable error range for the particular value.

[0017] Synthetic Peptides In some embodiments, the synthetic peptide produced by the methods of the present disclosure comprises the amino acid sequence of Cys1Cth2Glu3Leu4Cys5Cys6Asn7Val8Ala9Cys 10 Tyr 11 Gly 12 Cys 13 It can be represented linearly as (SEQ ID NO:1).

[0018] The synthetic peptide of SEQ ID NO:1 contains four cysteine ​​residues that form two disulfide bonds and a defined linkage (Cys1-Cys6, Cys5-Cys 13 ,Cth2-Cys 10 ) and a cystathione (Cth) unit (combining homocysteine ​​and cysteine, sharing a side chain sulfur atom) that provides an internal sulfide (or thioether) bond with the cystathione (Cth) unit.

[0019] For purposes herein, the two portions of the linear sequence are Cth2 and Cys 10and a thioether bond connects the carbons of the sulfur homocysteine ​​(Hcy) side chain and the des-SH cysteine ​​side chain; this double amino acid corresponds to a cystathionine (Cth) residue, but the proposed nomenclature facilitates interpretation when using the three-letter code designation of the residues; the peptide bond formed by the α-amino carboxyl group at position 1 is named "Cth" and the peptide bond formed by the α-amino carboxyl group at position 2 is named "Cys", see Scheme 1 above.

[0020] Alternatively, for purposes herein, the two moieties of the building block can be designated as [Hcy] and [Cys], respectively, with the sulfur of the homocysteine ​​(Hcy) side chain being shared with the side chain of cysteine ​​(Cys) to form a thioether bridge, and this double amino acid corresponding to the cystathionine (Cth) residue, although the proposed designation facilitates explanation when using the three-letter code designation of the residues. Using this alternative nomenclature, SEQ ID NO:1 can be represented as follows: Cys1Hcy2Glu3Leu4Cys5Cys6Asn7Val8Ala9Cys 10 Tyr 11 Gly 12 Cys 13 (Sequence number 1).

[0021] In some embodiments, Cth2-Cys 10 The designation, or any variation thereof, is meant to describe the linkage between the side chains of two non-consecutive amino acids of SEQ ID NO:1 that form a thioether bridge as shown below. [ka]

[0022] In some embodiments, Cth2-Cys 10 or any variation thereof, describes cystathionine forming a peptide bond at positions 2 and 10 of the synthetic peptide, forming a thioether bridge.

[0023] In some embodiments, the synthetic peptide of SEQ ID NO:1 has the formula: [ka] It can be expressed as:

[0024] Methods for Producing Synthetic Peptides The methods described herein begin by (i) chemically synthesizing a linear peptide C-terminally attached to a solid support using a plurality of amino acids and at least one polyamino acid synthon, the linear peptide having protecting groups at one or more amino acids and / or the polyamino acid synthon, in some embodiments, at least one amine group of the polyamino acid synthon has a different protecting group than the N-terminus of the linear peptide.

[0025] In some embodiments, the solid support is selected from the group consisting of Wang resin, trityl resin, and Rink resin.

[0026] In some embodiments, the solid support has a loading of about 0.10 mmol / g, about 0.20 mmol / g, about 0.30 mmol / g, about 0.40 mmol / g, about 0.50 mmol / g, about 0.60 mmol / g, about 0.70 mmol / g, about 0.80 mmol / g, about 0.90 mmol / g, or about 1.00 mmol / g. In some embodiments, the solid support has a loading of about 0.70 mmol / g. In some embodiments, the solid support has a loading of about 0.90 mmol / g.

[0027] In some embodiments, the polyamino acid synthon has the formula: [ka] (In the formula, P 1 and P 2 is hydrogen or an amine protecting group, with the proviso that P 1 and P 2 If both are amine protecting groups, then they are not the same amine protecting group, and P 3 is hydrogen or a carboxylic acid protecting group, P 4is hydrogen or a thiol protecting group.

[0028] In a preferred embodiment, P 1 and P 2 are different amine protecting groups, P 3 is a carboxylic acid protecting group, P 4 is a thiol protecting group.

[0029] In some embodiments, the protecting group is selected from the group consisting of fluorenylmethyloxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), carboxybenzyl (Cbz), trityl, methyl, ethyl, tert-butyl, allyl (All), 2,4-dimethoxybenzyl (Dmb), 9-fluorenylmethyl (Fm), benzyl (Bn), tert-butyldimethylsilyl, allyloxycarbonyl (alloc), tert-butyloxycarbonyl, acetamidomethyl (Acm), 3-nitro-2-pyridinesulfenyl (NPYS), and 2-pyridinesulfenyl (Pyr).

[0030] In some embodiments, the amine protecting group P 1 and P 2 are each selected from the group consisting of fluorenylmethyloxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), and carboxybenzyl (Cbz). 1 or P 2 is a tert-butyloxycarbonyl (Boc) protecting group. In some embodiments, P 1 or P 2 is a 9-fluorenylmethoxycarbonyl (Fmoc) protecting group. In some embodiments, P 1 is a tert-butyloxycarbonyl (Boc) protecting group, and P 2 is the 9-fluorenylmethoxycarbonyl (Fmoc) protecting group.

[0031] In some embodiments, the carboxylic acid protecting group P 3is selected from the group consisting of methyl, ethyl, tert-butyl, allyl (All), 2,4-dimethoxybenzyl (Dmb), 9-fluorenylmethyl (Fm), and benzyl (Bn). 3 is an allyl (All) protecting group.

[0032] In some embodiments, P 4 is a trityl protecting group.

[0033] In some embodiments, the subunits of the polyamino acid synthon have the D configuration, e.g., the synthon is a D-enantiomer. In some embodiments, a polyamino acid synthon having subunits in the D configuration can be represented by the formula: [ka]

[0034] In some embodiments, the subunits of the polyamino acid synthon have the L-configuration, e.g., the synthon is an L-enantiomer. In some embodiments, a polyamino acid synthon having subunits in the L-configuration can be represented by the formula: [ka]

[0035] In some embodiments, the subunits of the polyamino acid synthon have both the D- and L-configurations.

[0036] In some embodiments, the amino acid side chains of the linear peptide have a protecting group. In some embodiments, the amino acid side chain protecting group is selected from the group consisting of tert-butyl (tBu), trityl (Trt), allyl (All), cyclohexyl, 2-phenylisopropyl, acetamidomethyl (Acm), benzyl (Bzl), 4-methylbenzyl (4-MeBzl), 4-methoxybenzyl (4-MeOBzl), 9-fluorenylmethyl (Fm), tert-butylthio (t-buthio), 4-methoxytrityl (Mmt), xanthyl (Xan), 2,6-dichlorobenzyl (2,6-Cl2Bzl), and 2-bromobenzyl carbonate (2-BrZ). In some embodiments, the amino acid side chain protecting group is tert-butyl (tBu) or trityl (Trt).

[0037] In some embodiments, the amino acid side chains of the linear peptide having a protecting group at the side chain are Cys1, Glu3, Cys5, Cys6, Asn7, Tyr, 11 , and Cys 13 It is.

[0038] In some embodiments, the multiple amino acids and synthons are coupled by carbodiimide mediated reactions or with non-carbodiimide coupling agents such as 1-[bis(dimethylamino)methylene]-1H-1,2,3 triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 1H-benzotriazolium 1-[bis(dimethylamino)methylene]-5-chloro-hexafluorophosphate(1-),3-oxide (HCTU), O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), 1-[(1-(cyano-2-ethoxy-2-oxoethylideneaminooxy)-dimethylamino-morpholinomethylene)]methanaminium hexafluorophosphate (COMU), 1-cyano-2-ethoxy-2-oxoethylideneaminooxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyOxim), benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), 7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP), or propanephosphonic anhydride (T3P) mediated reaction to form the linear peptide of step (i).

[0039] In some embodiments, at least one amino acid from a plurality of peptides and / or synthons is coupled by a carbodiimide-mediated reaction to form the linear peptide of step (i). In some embodiments, the carbodiimide is selected from the group consisting of diisopropylcarbodiimide (DIC), dicyclohexylcarbodiimide (DCC), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). In some embodiments, the carbodiimide is DIC.

[0040] In some embodiments, the carbodiimide-mediated reaction mixture further comprises an amino acid racemization inhibitor. In some embodiments, the racemization inhibitor is selected from the group consisting of 2-hydroxypyridine-N-oxide (HOPO), 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azo-benzotriazole (HOAt), and 2-cyano-2-(hydroxyimino)acetate). In some embodiments, the racemization inhibitor is 2-cyano-2-(hydroxyimino)acetate.

[0041] In some embodiments, the solvent for the carbodiimide-mediated reaction is, but not limited to, N-methylpyrrolidinone (NMP), dichloromethane (DCM), chloroform, or dimethylformamide (DMF). In some embodiments, the solvent for the carbodiimide-mediated reaction is N-methylpyrrolidinone (NMP).

[0042] In some embodiments, pyridine is used with the carbodiimide-mediated reaction to prevent premature cleavage of the linear peptide from the solid support.

[0043] In some embodiments, at least one amino acid is coupled by a non-carbodiimide coupling agent, hi some embodiments, the non-carbodiimide coupling agent is TBTU.

[0044] The linear peptide of step (i) may be referred to in the present application as a "linear 12-mer" and has the following formula: [ka] (SEQ ID NO:2), in which the cystathionine thioether side chain bridge -CH-CH-S-CH- is [ka] In some embodiments, one or more of the underlined amino acids are side chain protected. In some embodiments, all of the underlined amino acids are side chain protected.

[0045] After the linear peptide is formed in step (i), the linear peptide is cleaved from the solid support in step (ii) to produce a protected peptide.

[0046] In some embodiments, the linear peptide is cleaved from the resin by treatment with a dilute acidic solution. In some embodiments, treatment with a dilute acidic solution preserves the side chain protecting groups and the protecting groups of the polyamino acid synthons. In some embodiments, the dilute acidic solution is, for example, dilute trifluoroacetic acid (TFA) or dilute bromotrimethylsilane (TMSBr). In some embodiments, the dilute acidic solution is a TFA solution. In some embodiments, the dilute acidic solution is a 1% trifluoroacetic acid (TFA) in dichloromethane (DCM) solution.

[0047] Following cleavage of the linear peptide from the resin to generate the protected peptide, an amino acid having an unprotected amine group, a protected carboxylic acid group, and optionally a protected amino acid side chain, is coupled to the C-terminus of the protected peptide in step (iii).

[0048] In some embodiments, the protected peptide obtained in step (ii) is used directly in step (iii).

[0049] In some embodiments, the amino acid attached to the C-terminus of the protected peptide is cysteine. In some embodiments, the cysteine ​​is protected. In some embodiments, the amino acid attached to the C-terminus is S-trityl-L-cysteinyl-Ot-butyl-ester. In some embodiments, the protected peptide obtained in step (ii) has the following formula: [ka] It can be represented by:

[0050] (SEQ ID NO:3) In some embodiments, one or more of the underlined amino acids have their side chains protected. In some embodiments, all of the underlined amino acids have their side chains protected.

[0051] Following coupling of the amino acid to the C-terminus in step (iv), one amine protecting group and one carboxylic acid protecting group are removed from the protected peptide formed in step (iii) to form a partially unprotected peptide having an unprotected amine and an unprotected carboxylic acid group.

[0052] In some embodiments, the carboxylic acid that is deprotected in step (iv) is derived from a protected carboxylic acid group of a polyamino synthon.

[0053] Once the protecting groups are removed, in step (v), the unprotected amine and the unprotected carboxylic acid groups are coupled to form the cyclized peptide.

[0054] Following formation of the cyclized peptide in step (v), the cyclized peptide is globally deprotected in step (vi) to obtain a globally deprotected peptide. In some embodiments, the global deprotection step (vi) comprises the addition of a cocktail comprising at least ammonium iodide (NH4I) and thioanisole.

[0055] Once the cyclized peptide has been globally deprotected, in step (vii), the peptide folds back to form one or more additional bridges, forming Cys1Cth2Glu3Leu4Cys5Cys6Asn7Val8Ala9Cys 10 Tyr 11 Gly 12 Cys 13 A synthetic peptide of (SEQ ID NO: 1) is obtained.

[0056] In some embodiments, the cross-linked synthetic peptide formed in step (vii) is a nucleotide sequence consisting of the following amino acid residues of the synthetic peptide: Cys1 and Cys6, Cth2 and Cys 10 , and Cys5 and Cys 13 In some embodiments, the covalent bond between Cys1 and Cys6 and between Cys5 and Cys 13 In some embodiments, the covalent bond between Cth2 and Cys is a disulfide bond. 10 The covalent bond between is a thioether bond.

[0057] Following folding of the globally deprotected peptide, the synthetic peptide of SEQ ID NO:1 is purified.

[0058] In some embodiments, the method includes an optional step (viii) of modifying the N-terminus of the synthetic peptide with one or more chemical moieties. In some embodiments, the N-terminus of the synthetic peptide is modified with an acetyl group. When the N-terminus of the synthetic peptide is modified with one or more chemical moieties, the synthetic peptide is purified twice, once immediately after the folding step (vii) and once after the N-terminal modification.

[0059] In some embodiments, the method further comprises precipitating the synthetic peptide from the solution. In some embodiments, the precipitation step comprises an acidification step followed by dilution with an organic solvent mixture. In some embodiments, the organic solvent mixture comprises at least one of acetonitrile or methyl tert-butyl ether (MTBE).

[0060] Formula I: [ka] Also described herein is a method for preparing a synthetic peptide of the formula: (i) C-terminal resin-bound Tyr-Gly peptide (Tyr amino acid residue is protected) is reacted with a carboxyl group of formula II: [ka] (In the formula, P 1 and P 2 are different amine protecting groups, P 3 is a carboxylic acid protecting group, P 4 is a thiol protecting group) to a polyamino acid synthon of formula III: [ka] coupling a C-terminal resin-bound Tyr-Gly peptide to a polyamino acid synthon of formula II to form a resin-bound peptide of formula II; (ii) P of formula III 2 The protecting groups are removed to give a compound of formula IV having an unprotected amine group: [ka] and obtaining a resin-bound peptide of (iii) P via a free amine group of formula IV 2 -alanine to the resin-bound peptide of formula IV to obtain a peptide of formula V: [ka] forming a resin-bound peptide of (iv) P in formula V 2 The protecting group is removed to give the free amine group, which is then converted to P 2 coupling to an amino acid, coupling the amino acid, the side chain of which may be protected; (v) Step (iv) is repeated six more times to obtain a compound of formula VI: [ka] forming a resin-bound peptide of forming a cycloalkyl group having at least one amino acid side chain protected; (vi) cleaving the peptide of formula VI from the resin to form a linear peptide having a C-terminal carboxylic acid group; (vii) coupling the C-terminal carboxylic acid group of the linear peptide to the amine group of a cysteine ​​to obtain a protected peptide of formula VII, the cysteine ​​contains a carboxylic acid protecting group; The side chain of the cysteine ​​amino acid may be protected. [ka] (In the formula, P 5 , P 3 (wherein the protecting group is a carboxylic acid group different from (viii)P 2 Protecting Groups and P 3 removing the protecting groups to provide a free amine group and a free carboxylic acid group; (ix) coupling a free amine group with a free carboxylic acid group to form a compound of formula VIII: [ka] and obtaining a cyclized peptide of the formula: (x) globally deprotecting the cyclized peptide to obtain a globally deprotected peptide; (xi) folding the globally deprotected peptide by forming two disulfide bonds to obtain the synthetic peptide of formula I.

[0061] As used herein, P 2 -Amino acids are P 1 The amine protecting group is an amino acid whose amine group is protected by an amine protecting group different from the amine protecting group of 2 Alanine has the following structural formula: [ka] would have.

[0062] In some embodiments, the method comprises acetylating a free amine group of formula I to form formula IX: [ka] The method further comprises obtaining a synthetic peptide of the formula:

[0063] In some embodiments, P 1 is an acetyl group, and steps (i) to (ix) are carried out as described above. However, in step (x) during the overall deprotection process, P 1 The acetyl group represented by formula (IX) is not removed and step (x), the folding step, forms a compound represented by formula IX.

[0064] In some embodiments, the Glu, Cys, Cys, Asn, Gly and Cys residues of formula VII have side chain protecting groups. In some embodiments, the amino acid side chain protecting group is selected from the group consisting of tert-butyl (tBu), trityl (Trt), allyl (All), cyclohexyl, 2-phenylisopropyl, acetamidomethyl (Acm), benzyl (Bzl), 4-methylbenzyl (4-MeBzl), 4-methoxybenzyl (4-MeOBzl), 9-fluorenylmethyl (Fm), tert-butylthio (t-buthio), 4-methoxytrityl (Mmt), xanthyl (Xan), 2,6-dichlorobenzyl (2,6-Cl2Bzl), and 2-bromobenzyl carbonate (2-BrZ). In some embodiments, the amino acid side chain protecting group is tert-butyl (tBu) or trityl (Trt).

[0065] In some embodiments, P 1 and P 2 Each of P is a protecting group selected from the group consisting of fluorenylmethoxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), carboxybenzyl (Cbz), and allyloxycarbonyl (Alloc). 1 is a tert-butyloxycarbonyl (Boc) protecting group. In some embodiments, P 2 is a fluorenylmethoxycarbonyl (Fmoc) protecting group.

[0066] In some embodiments, P 3 is a protecting group selected from the group consisting of methyl, ethyl, tert-butyl, allyl (All), trityl, 2,4-dimethoxybenzyl (Dmb), 9-fluorenylmethyl (Fm), and benzyl (Bn). 3 is an allyl (All) protecting group.

[0067] In some embodiments, P 4 is a protecting group selected from the group consisting of acetamidomethyl (Acm), tert-butyl (t-Bu), 3-nitro-2-pyridinesulfenyl (NPYS), 2-pyridinesulfenyl (Pyr), and trityl (Trt). 4 is a trityl protecting group. In some embodiments, P 4 is a tert-butyl protecting group.

[0068] compound

[0069] Also, the following structural formula: [ka] (In the formula, P 1 and P 2 is hydrogen or an amine protecting group, with the proviso that P 1 and P 2 are amine protecting groups, they are not the same amine protecting group, P 3 is hydrogen or a carboxylic acid protecting group, P 4 is hydrogen or a thiol protecting group. Disclosed herein is a compound represented by:

[0070] In some embodiments, P 1 , P 2 , P 3 , and / or P 4At least one of P is hydrogen. 1 ~P 4 are hydrogen.

[0071] In some embodiments, P 1 ~P 4 are each a protecting group (e.g., P 1 and P 2 are amine protecting groups, and P 3 is a carboxylic acid protecting group, P 4 is a thiol protecting group).

[0072] In some embodiments, P 1 and P 2 are each an amine protecting group selected from the group consisting of acetyl, fluorenylmethoxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), carboxybenzyl (Cbz), and allyloxycarbonyl (Alloc). 1 is an acetyl group. In some embodiments, P 1 is a tert-butyloxycarbonyl (Boc) protecting group. In some embodiments, P 2 is the Fmoc protecting group.

[0073] In some embodiments, P 3 is a carboxylic acid protecting group selected from the group consisting of methyl, ethyl, tert-butyl, allyl (All), trityl, 2,4-dimethoxybenzyl (Dmb), 9-fluorenylmethyl (Fm), and benzyl (Bn). 3 is an allyl (All) protecting group.

[0074] In some embodiments, P 4 is a thiol protecting group selected from the group consisting of acetamidomethyl (Acm), tert-butyl (t-Bu), 3-nitro-2-pyridinesulfenyl (NPYS), 2-pyridinesulfenyl (Pyr), and trityl (Trt).4 is a trityl protecting group.

[0075] In some embodiments, the compound or a pharma- ceutically acceptable salt thereof has formula A: [ka] It is a compound of the formula:

[0076] In some embodiments, to arrive at a compound of formula A, three building blocks are synthesized separately (Parts A-C) and then combined (Part D) to synthesize a compound of formula A, as shown in the following scheme: Part A: [ka] Part B: [ka] Part C: [ka] Part D: [ka] [ka]

[0077] In some embodiments, to arrive at compounds of formula A, the diblock compound (Alloc-HCys((Fmoc-Ala-OH)3-yl)-OAll) is first synthesized as shown in the following scheme: [ka] EXAMPLES

[0078] The following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention in any way, since numerous variations and equivalents encompassed by the present invention will become apparent to those of skill in the art upon reading this disclosure.

[0079] Reagents and solvents

[0080] The starting materials, reagents and solvents used in the preparation of the claimed peptides are listed in Tables 1-3, respectively. [Table 1-1] [Table 1-2] [Table 1-3] [Table 2] [Table 3]

[0081] Example 1 Process for producing synthetic peptide of SEQ ID NO:1 Introduction The peptide of SEQ ID NO:1, the N-terminally modified peptide of SEQ ID NO:1, or a pharma- ceutically acceptable salt thereof will be used in the planned clinical trials and was manufactured in accordance with Good Manufacturing Practice (GMP) regulations. All abbreviations are listed in Table 2 (list of reagents) and Table 3 (list of solvents), respectively.

[0082] The peptide of SEQ ID NO:1, the N-terminal modified peptide of SEQ ID NO:1 or a pharma- ceutically acceptable salt thereof was prepared according to the synthesis scheme described below. The synthetic route combined the stepwise synthesis of parts of the peptide chain according to well-established principles of solid-phase peptide chemistry. This was followed by successive steps to incorporate the C-terminal amino acid, cyclization to form a thioether bridge, folding, primary purification, N-terminal acetylation, final purification by preparative-scale chromatography, and isolation of the drug substance as a solid by precipitation.

[0083] An exemplary manufacturing process for the peptide of SEQ ID NO:1, the N-terminal modified peptide of SEQ ID NO:1, or a pharma- ceutically acceptable salt thereof is shown in Figure 1. All starting materials constituting the peptide primary sequence are introduced in a protected form compatible with the selected chemistry. All optically active amino acid residues are used in the naturally occurring "L" form.

[0084] Folding leading to two disulfide bridges between the designated cysteine ​​residues is induced after completion of the assembly. A thioether bond between the homocysteine ​​side chain (at position 2) and the cysteine ​​side chain (at position 10) is preformed in the tripeptide building block. Subsequent cyclization is induced by the formation of a lactam bond as described below (step 5). After final chromatographic purification, the drug substance is isolated by precipitation and drying.

[0085] Peptide synthesis Step 1: Stepwise solid-phase assembly

[0086] The primary amino acid sequence was determined by dividing the glycine residues (Gly in synthetic peptides) 12 ) was constructed by an iterative process starting from preloaded 2-chlorotrityl resin. Nine successive cycles, detailed in Tables 4-5, were performed as follows: - Removal of the N-terminal Fmoc protecting group from the previously introduced amino acid residue by treatment with base (piperidine) in dimethylformamide (DMF) followed by extensive washing with DMF. This deprotection was not carried out after the last incorporation step (glutamic acid). The deprotection reaction was monitored by HPLC. -Coupling of the next Fmoc-protected amino acid or building block (suitably protected on its side chain as relevant) in NMP by the action of DIC in the presence of Oxyma®, except for the first reaction (coupling of tyrosine to resin-immobilized glycine) which is carried out in NMP by the action of TBTU in the presence of DIPEA. The coupling reactions were monitored by HPLC. - Thorough washing with DMF. [Table 4] [Table 5]

[0087] Coupling

[0088] NMP was used as a solvent for dissolving the amino acids, the DeFmoc solution, the coupling agent, the capping solution and the pyridine-kick. The pyridine-kick is the addition of 3.6 equivalents of pyridine per coupling cycle to avoid partial cleavage of the growing peptide from the solid support. The pyridine-kick was used in all coupling reactions except when coupling cysteine ​​residues as discussed below. DMF was used only for the washing steps after the coupling and Defmoc steps. Because NMP was used instead of DMF, the coupling times had to be extended from 90 to 180 min for standard amino acids and from 180 to 360 min for tripeptide building blocks. Only 1.2 equivalents of tripeptide were used instead of 2.0 equivalents for standard coupling. The conditions for the standard process are shown in Table 4. SPPS of the protected linear peptides was carried out on a 0.75 mmol scale on a Tribute automated synthesizer using pre-packed H-Gly-CT resin as described in the protocol (Table 5). The pyridine kick was used to minimize premature cleavage of the peptide from the resin due to the acidic coupling conditions.

[0089] Analytical HPLC LC-MS analysis does not show any hints for a high content of deleted sequences. The experiment showed that 1.2 equivalents of the tripeptide building block were suitable for complete coupling.

[0090] Cysteine ​​racemization (pyridine kick)

[0091] SPPS using DIC / Oxyma resulting in acidic coupling conditions can cause partial cleavage of the peptide from the resin. Therefore, the pyridine-kick was established. As mentioned above, the pyridine-kick is the addition of a portion of pyridine to drive the reaction to completion. It is known that racemization can occur for the elementary coupling of cysteines. Therefore, regarding the risk of racemization, the pyridine-kick is not performed during the coupling of cysteine ​​residues, since cysteine ​​residues are particularly sensitive to alkaline conditions (e.g. induced by pyridine). There was none Therefore, test peptides were synthesized once with and once without a pyridine kick, using methionine instead of the L-tripeptide for cysteine-coupling.

[0092] The use of pyridine kick for all couplings resulted in a D-cysteine ​​content of 1.49% compared to 1.35% for peptides synthesized without pyridine kick for cysteine ​​couplings. Also, there was no effect on the resin yield for SPPS. In conclusion, there was a minimal effect on the yield and quality of the obtained product. However, to reduce the risk of racemization, the pyridine kick was not used for cysteine ​​couplings and tripeptide (chemically similar to the protected cysteine) couplings.

[0093] Resin Filling Amount

[0094] The effect of resin loading was investigated while generating new material for downstream processing. In one experiment, preloaded H-Gly-CT resin with a loading of 0.7 mmol / g was used, while in another experiment, preloaded H-Gly-CT resin with a loading of 0.9 mmol / g was used. The crude peptide shows the same quality and yield on the resin.

[0095] SPPS Run

[0096] The assembly was carried out on a 7.5 mmol scale on 0.65 mmol / g preloaded H-Gly-2CT resin. Each coupling step was monitored by Kaiser test. The results are shown in Table 6. A mild test cleavage was performed and analyzed by UHPLC. A purity of 87.3% AN was reached. The yield on resin was 98%.

[0097] A second SPPS batch was run automatically. The scale was again 7.5 mmol, with a resin loading of 0.69 mmol / g. A purity of 89.4% AN was achieved with a quantitative (>=100%) yield on resin. [Table 6]

[0098] Step 2: Cleavage of the resin-peptide bond

[0099] The peptide is cleaved from the resin under mild acidic treatment that preserves the side chain protecting group of the Cys1 residue as well as the N-alpha Boc protecting group. The completion of this reaction is time specific. The reaction mixture is concentrated by evaporation and the solvent is exchanged into DMF.

[0100] To avoid the formation of a rubbery solid that adheres to the glassware, direct coupling of a C-terminal cysteine ​​was tested. This eliminated one isolation step and reduced cycle time. Direct coupling was tested in a mild cleavage batch, where different anti-solvents were already tested to precipitate the peptide. The batch was evaporated to oil, reconstituted in DCM, and evaporated again to remove all residual anti-solvent. After reconstitution in DCM, the peptide was used directly for cysteine ​​coupling. Table 7 shows the cleavage conditions.

[0101] [Table 7]

[0102] Step 3: Incorporation of the C-Terminal Residue and In-Situ Deprotection of the Protecting Group from Glu3

[0103] The cleaved peptide solution from step 2 was activated by DIC / HOPO and coupling with H-Cys(Trt)-OtBu was carried out in DMF in the presence of DIEA as a base. Upon completion of the reaction, monitored by HPLC, the Fmoc protecting group of the N-αGlu3 residue was cleaved by adding piperidine directly to the reaction mixture. Upon completion of the reaction, monitored by HPLC, the product of the reaction was isolated by extraction, precipitation, filtration and drying. Table 8 shows the cysteine ​​coupling conditions. [Table 8]

[0104] Step 4: Removal of protecting groups from Hcy2

[0105] The O-allyl ester protecting group of C-alpha-Hcy2 was then removed by dissolving it in DCM and cleaving the O-allyl ester with a palladium-containing catalyst (Pd(PPh3)4) in the presence of phenylsilane as a scavenger. The progress of the reaction was monitored by HPLC. The DeAllyl conditions were as follows: 1) Dissolve Pd(PPh3)4 (0.05 equivalents) in DCM. 2) Add phenylsilane (2 eq.) and 13mer to a Pd(PPh3)4 / DCM solution. 3) Stir for 1 hour and monitor the reaction progress by HPLC. 4) Once the deallylation is complete, the solution is ready for the next step.

[0106] Step 5: Cyclization by Hcy2→Glu3 coupling

[0107] Upon completion of step 4, HOPO and DIC were added directly to the reaction mixture to induce cyclization by coupling between the carboxylic acid functional group of Hcy2 and the amine functional group of Glu3. The reaction was monitored by HPLC. Table 9 shows the cyclization conditions.

[0108] For all cyclization concentrations (5, 10, 25 or 50 mg / mL), no oligomeric peaks were visible in the chromatograms. All four experiments were performed at the same scale, and the crude yield for each experiment was 40 ± 2 mg. The maximum peak area was obtained for cyclization at 50 mg / mL, indicating that the crude contained the maximum amount of peptide.

[0109] Cyclization was also tested at 100 mg / mL. To achieve this, the coupling agent was added directly to the deallylation solution. As judged from the chromatogram, no peaks are seen that indicate oligomerization. Both cyclization concentrations (50 mg / mL and 100 mg / mL) give very similar results. The higher concentration did not provide any benefit in terms of purity. [Table 9]

[0110] Step 6: Global deprotection

[0111] Pd removal

[0112] Upon completion of the cyclization, the reaction mixture was concentrated by evaporation under vacuum (to about 50% of the original volume) and treated with Si-thiol, a scavenger specific for the removal of Pd-based catalysts. The complexed Pd scavenger was filtered off and the collected filtrate was further concentrated by evaporation. Table 10 shows the Pd removal conditions. [Table 10]

[0113] The still protected cyclized 13mer peptide was then subjected to complete deprotection by addition of a mixture of DTT, water, thioanisole, TIS and NH4I in TFA. Upon completion of the reaction, the crude deprotected peptide was recovered by precipitation in n-heptane and MBTE. The resulting solid was dried under vacuum. Table 11 shows the overall deprotection conditions. [Table 11]

[0114] Step 7: Folding via disulfide bridges

[0115] The crude deprotected peptide from step 6 was added to ammonium bicarbonate and DMSO was added to deprotect the Cys1 and Cys6, and Cys5 and Cys7, respectively. 13 Folding was induced by the formation of two disulfide bridges between the side chains of . The folding reaction was monitored by HPLC. Table 12 shows the conditions used for the folding process. [Table 12]

[0116] Step 8: Primary (first) purification

[0117] Upon completion of step 7, the reaction mixture was acidified with TFA, Celite was added as a filter aid, and the resulting slurry was filtered. The clear filtrate was loaded directly onto a preparative HPLC column packed with a C18(3) stationary phase. Purification was performed by gradient elution at 0.1% TFA in acetonitrile and 0.1% TFA in acetonitrile:water (5:95 v / v). Individual fractions were collected and selection was based on analytical HPLC monitoring: fractions showing a purity of ≧90% (area %) were pooled.

[0118] Step 9: N-Terminal Acetylation

[0119] Direct acetylation of the folded peptide in the folding solution was tested. After adding 15 equivalents of AcOSu, very little conversion was detected. After pH adjustment and additional AcOSu charge, still no substantial conversion was obtained. This was repeated with a second folding solution with the same results. Addition of Ac2O did not result in better conversion.

[0120] To gain insight into the acetylation reaction of this compound, two experiments were carried out overnight using 15 equivalents of AcOSu and 2 equivalents of Ac2O, respectively. Both reactions showed promising results, with the reaction with AcOSu not going to completion after 15 hours.

[0121] The reaction was repeated using increased amounts of AcO, 3 and 4 equivalents, respectively, and monitored after 18 hours. The larger amount of acetylating agent appeared to drive the reaction to completion, and shortening the reaction time appeared to reduce the amount of side reactions, especially for the two largest impurities that elute late.

[0122] Therefore, the final process was determined to be carried out with 4.0 equivalents of Ac2O. Therefore, the acetylation step 9 was carried out from the pool collected from the primary purification step 8 diluted in ammonium bicarbonate solution containing DMSO, and then the N-αCys1 residue was acetylated with acetic anhydride. The reaction was monitored by HPLC. Table 13 shows the conditions for the acetylation step. [Table 13]

[0123] Step 10: Secondary purification

[0124] The acetylation reaction mixture was subjected to final purification by diluting 1 / 1 with ammonium acetate solution, adjusting the pH to 7-8 with 30% (v / v) aqueous ammonium hydroxide, and injecting into a preparative HPLC column packed with a C18(3) stationary phase. Purification was performed by gradient elution with acetonitrile and 25 mM aqueous ammonium acetate:acetonitrile (95:5, v / v).

[0125] Individual fractions were collected and the selection was based on analytical HPLC monitoring: fractions showing a purity of 95% (area %) or greater and no single impurity greater than 1.0% (area %) were pooled.

[0126] A second purification, with acetylation conditions optimized to reduce starting material to less than 0.1%, provided slight polishing of the material and promoted ion exchange from trifluoroacetate to acetate.

[0127] Step 11: Precipitation, isolation by filtration and drying

[0128] The purified pool collected from the secondary purification step was concentrated by evaporation under vacuum to a target concentration of 50-100 mg / mL. The product was then precipitated by acidification (acetic acid), dilution with acetonitrile, and addition of MBTE. The precipitate was collected through a 0.2 μm filter and washed extensively with MBTE / acetonitrile solution. Conditions are shown in Table 14.

[0129] The solid was isolated by wetting with 30% (v / v) aqueous acetonitrile followed by water and final drying under vacuum to give the synthetic peptide of SEQ ID NO:1. [Table 14]

[0130] Example 2 Preparation of Alloc-HCys(Fmoc-Ala-OH)-3-yl)-OAll The following reaction scheme illustrates the preparation of the title compound. [ka]

[0131] Synthesis of Alloc-Hser-OAll [ka]

[0132] In a 5 L three-neck flask, 500 g of (S)-3-aminodihydrofuran-2(3H)-one hydrobromide was dissolved in 1 L of water. A solution of 230 g of NaOH in 1 L of water was added. The reaction was monitored by TLC. After completion of the reaction, concentrated HCl was added to adjust the pH to 7.5. 230 g of Na2CO3 was added and the mixture was cooled to 0°C. 331 g of alloxycarbonyl chloride (Alloc-Cl) was added dropwise while maintaining the temperature at 0-10°C and the pH at 7.5. The mixture was stirred overnight. The solvent was then removed and then 2500 mL of DMF was added. 230 g of NaHCO3 was added followed by 665 g of allyl bromide. The mixture was stirred overnight and then 7000 mL of water was added to quench the reaction. The mixture was extracted with MTBE (5 L x 3), and the combined organic phase was washed successively with 800 mL of NaHCO3, 800 mL of KHSO4, and 800 mL of brine. After removing the solvent MTBE, the residue was purified by column chromatography (SiO2) to give 273.9 g oily product. 1 H NMR (400 MHz, chloroform-d) δ 6.02 - 5.65 (m, 3H), 5.39 - 5.07 (m, 4H), 4.58 - 4.17 (m, 4H), 3.80 - 3.52 (m, 2H), 3.08 (s, 1H), 2.77 - 2.17 (m, 1H), 2.17 - 2.01 (m, 1H), 1.88 - 1.64 (m, 1H). ESI-MS C11H17NO5 [M+H] + Calculated value: 244.12; Measured value: 244.08.

[0133] Synthesis of Alloc-Abu(4-Br)-OAll [ka]

[0134] In a 10 L three-neck flask, 378 g Alloc-HSer-OAll and 216 g Et3N were dissolved in 3400 mL DCM. Then, 215 g mesyl chloride (MsCl) was added dropwise while maintaining the temperature at 0-10 °C. After the reaction was completed, 3400 mL acetone was added, followed by 1356 g LiBr in small portions. The reaction was maintained at 25-30 °C overnight. 200 g LiBr and 520 mL acetone were added to complete the reaction. The solvent was then removed and the residue was combined with another batch starting with 153 g Alloc-HSer-OAll. 572 g Alloc-Abu(4-Br)-OAll was obtained after column chromatography (SiO2). 1 H NMR (400 MHz, chloroform-d) δ 5.99 - 5.75 (m, 2H), 5.52 (d, J = 8.2 Hz, 1H), 5.40 - 5.11 (m, 4H), 4.63 (d, J = 5.8 Hz, 1H), 4.55 (d, J = 5.4 Hz, 2H), 4.48 (td, J = 8.4, 5.1 Hz, 1H), 3.42 (t, J = 7.0 Hz, 2H), 2.50 - 2.34 (m, 1H), 2.33 - 2.13 (m, 1H). ESI-MS C11H16BrNO4 [M+H] + Calculated value: 306.03; Measured value: 306.11.

[0135] Synthesis of (Fmoc-Cys-O(t-Bu))2 [ka]

[0136] 210 g of L-Cystine and 4 kg of t-butyl acetate were mixed in a 5 L three-neck flask. 301.2 g of HClO4 were added dropwise. The mixture was stirred overnight. 2.3 kg of K2CO3 and 4.0 kg of water were added. The mixture was stirred overnight and then filtered through 20 g of Celite. After separation, the organic layer was washed with 1 L of brine and then concentrated to obtain 258.8 g of oily residue. The residue was dissolved in 2400 L of THF, after which 430 g of Fmoc-OSu was added. Then, 126 g of N-methylmorphine was added dropwise. The reaction mixture was stirred for 2 hours. The solvent of THF was removed and 1500 mL of DCM was added to dissolve the residue. The organic solution was washed successively with 10% citric acid solution, saturated NaHCO3, and brine. After removing the solvent, 500 mL of EtOAc was added. The mixture was slurred for 2 hours and then filtered. The filter cake was dried to give 453.3 g of (Fmoc-Cys-O(t-Bu))2. The yield was 65%. 1 H NMR (400 MHz, chloroform-d) δ 7.75 (d, J = 7.5 Hz, 2H), 7.60 (d, J = 7.5 Hz, 2H), 7.39 (t, J = 7.3 Hz, 2H), 7.29 (t, J = 7.2 Hz, 2H), 5.79 (d, J = 7.8 Hz, 1H), 4.74 - 4.53 (m, 1H), 4.50 - 4.29 (m, 2H), 4.29 - 4.17 (m, 1H), 3.37 - 3.07 (m, 2H), 1.50 (s, 9H).

[0137] Synthesis of Fmoc-Cys-O(t-Bu) [ka]

[0138] To a solution of 367 g of (Fmoc-Cys-O(t-Bu))2 in 4300 mL of THF was added 170 mL of (t-Bu)3P. The mixture was stirred for 2 h, and then 550 mL of water was added to quench the reaction. The mixture was stirred overnight. The THF solvent was removed and 1500 mL of EtOAc was added. The mixture was stirred for 0.5 h, and the organics were washed successively with 450 mL of 10% citric acid and 450 mL of brine. The organic solution was used directly in the next step.

[0139] Synthesis of Alloc-HCys((Fmoc-Ala-O(t-Bu))-3-yl)-OAll [ka]

[0140] In a 10 L 4-neck flask, 1080 g of tetrabutylammonium bromide was dissolved in 3350 mL of saturated NaHCO3. A 500 mL solution of 257 g of Alloc-Abu(4-Br)-OAll in EtOAc and 2.8 L of Fmoc-Cys-O(t-Bu) solution obtained from the last step were added. The mixture was stirred overnight. After washing with 1 L of brine, the organic solution was concentrated. Column chromatography (SiO2) gave 418 g of oily residue. The yield was 80%. 1H NMR (400 MHz, クロロホルム-d) δ 7.76 (d, J = 7.5 Hz, 2H), 7.61 (d, J = 7.5 Hz, 2H), 7.40 (t, J = 7.4 Hz, 2H), 7.31 (t, J = 7.4 Hz, 2H), 5.97 - 5.81 (m, 2H), 5.70 (d, J = 7.8 Hz, 1H), 5.43 (d, J = 8.3 Hz, 1H), 5.37 - 5.12 (m, 4H), 4.63 (d, J = 5.8 Hz, 1H), 4.61 - 4.43 (m, 4H), 4.39 (d, J = 7.2 Hz, 2H), 4.23 (t, J = 7.1 Hz, 1H), 4.12 (q, J = 7.1 Hz, 1H), 3.08 - 2.85 (m, 2H), 2.72 - 2.47 (m, 2H), 2.22 - 2.07 (m, 1H), 2.02 - 1.86 (m, 1H), 1.49 (s, 9H). ESI-MS C33H40N2O8S[M+Na] + についてのCalculated value: 647.24; Measured value: 647.24.

[0141] Synthesis of Alloc-HCys((Fmoc-Ala-OH)-3-イル)-OAll

change

[0142] A 5 L flask was charged with 308 g of Alloc-HCys((Fmoc-Ala-O(t-Bu)-3-yl)-OAll, 2430 mL of TFA and 53 g of i-PrSiH. The solution was stirred overnight at 20-25 °C, after which the solvent was removed. 520 mL of DCM was added and the solution was concentrated to remove the remaining TFA. 520 mL of DCM was added and the solution was concentrated to remove the remaining TFA. 1 L of MTBE was added to dissolve the residue. The organic phase was cooled to 37 °C and then cooled to 37 °C. The solution was neutralized with 4 L of saturated aqueous NaHCO3. After the mixture was filtered, the solid was dissolved in 3 L of DCM. The organic solution was acidified by washing with 1 L of 20% citric acid solution. The obtained organic solution was washed successively with 900 mL of 10% citric acid solution and 500 mL of brine. The organic layer was dried over 150 g of MgSO4, and then the mixture was filtered. The solvent was removed and the residue was stirred to solidify, giving 237 g of solid product. The yield was 84.4%. 1 H NMR (400 MHz, chloroform-d) δ 7.75 (d, J = 7.5 Hz, 2H), 7.60 (d, J = 7.5 Hz, 2H), 7.39 (t, J = 7.5 Hz, 2H), 7.30 (t, J = 7.4 Hz, 2H), 6.43 (s, 3H), 5.99 - 5.79 (m, 3H), 5.55 (d, J = 8.3 Hz, 1H), 5.39 - 5.13 (m, 4H), 4.76 - 4.28 (m, 8H), 4.23 (dd, J = 7.0 Hz, 1H), 3.22 - 2.76 (m, 2H), 2.74 - 2.48 (m, 2H), 2.14 (s, 1H), 1.97 (s, 1H). ESI-MS C29H32N2O8S [M+H] + Calculated value: 569.20; Measured value: 569.12.

[0143] Example 3 Preparation of Fmoc-L-Cth[3-Boc-L-Cys(Trt),4-O-allyl]-OH building block To arrive at the Fmoc-L-Cth[3-Boc-L-Cys(Trt),4-O-allyl]-OH building block, three building blocks are synthesized separately (parts A–C) and then combined (part D) to synthesize the title compound, as shown in the scheme below. Part A: [ka] Part B: [ka] Part C: [ka] Part D: [ka] [ka]

[0144] Other embodiments The scope of the present invention is not limited by the specific embodiments described herein. Indeed, various modifications of the present invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to be included within the scope of the appended claims. It should be further understood that all values ​​are approximate and are provided for illustrative purposes.

[0145] All patents, patent applications, publications, product descriptions, and protocols cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties for all purposes.

Claims

1. 1. A method for producing a synthetic peptide, or a pharmaceutically acceptable salt thereof, said method comprising: (i) chemically synthesizing a linear peptide comprising a plurality of amino acids and at least one polyamino acid synthon, said linear peptide having a C-terminus attached to a solid support; one or more amino acids of said plurality of amino acids, said at least one polyamino acid synthon, or a combination thereof, comprises a protecting group; the at least one polyamino acid synthon comprises at least one amine group having a protecting group different from that of the N-terminus of the linear peptide; (ii) cleaving the linear peptide from the solid support to produce a protected peptide; (iii) coupling an amino acid to the C-terminus of the protected peptide, the amino acid having an unprotected amine group, a protected carboxylic acid group, and an optionally protected amino acid side chain; (iv) removing one amine protecting group and one carboxylic acid protecting group from the protected peptide to form a partially unprotected peptide having an unprotected amine and an unprotected carboxylic acid group; (v) coupling the unprotected amine with the unprotected carboxylic acid group to form a cyclized peptide; (vi) globally deprotecting the cyclized peptide to obtain a globally deprotected peptide; (vii) folding the globally deprotected peptide to form one or more additional cross-links to obtain the synthetic peptide; (viii) optionally modifying the N-terminus of said synthetic peptide with one or more chemical moieties; (ix) purifying the synthetic peptide; The synthetic peptide has the amino acid sequence: Cys １ Cth ２ Glu ３ Leu ４ Cys ５ Cys ６ Asn ７ Val ８ Ala ９ Cys １０ Tyr １１ Gly １２ Cys １３ (SEQ ID NO: 1), 10. The synthetic peptide, comprising the following amino acid residues of the synthetic peptide: a) Cys １ and Cys ６ Between b) Cth ２ and Cys １０ Between and c) Cys ５ and Cys １３ The method of claim 1, wherein the covalent bond between

2. 10. The method of claim 1, further comprising precipitating the synthetic peptide from solution via acidification followed by dilution with an organic solvent mixture.

3. 2. The method of claim 1, wherein the solid support is selected from the group consisting of Wang resin, trityl resin, and Rink resin.

4. 10. The method of claim 1, wherein the solid support has a loading of about 0.10 mmol / g, about 0.20 mmol / g, about 0.30 mmol / g, about 0.40 mmol / g, about 0.50 mmol / g, about 0.60 mmol / g, about 0.70 mmol / g, about 0.80 mmol / g, about 0.90 mmol / g, or about 1.00 mmol / g.

5. The polyamino acid synthon has the formula: 【Chemistry 41】 (In the formula, P １ and P ２ are each an amine protecting group, P 1 and P 2 are not the same, P ３ is a carboxylic acid protecting group, P ４ is a thiol protecting group) The method of claim 1, wherein the compound is represented by

6. 2. The method of claim 1, wherein the protecting group is selected from fluorenylmethyloxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), carboxybenzyl (Cbz), trityl, methyl, ethyl, tert-butyl, allyl, 2,4-dimethoxybenzyl (Dmb), 9-fluorenylmethyl (Fm), benzyl (Bn), tert-butyldimethylsilyl, allyloxycarbonyl (alloc), tert-butyloxycarbonyl, acetamidomethyl (Acm), 3-nitro-2-pyridinesulfenyl (NPYS), or 2-pyridinesulfenyl (Pys).

7. P １ is tert-butyloxycarbonyl (Boc), and P ２ The method of claim 6, wherein is 9-fluorenylmethoxycarbonyl (Fmoc), P 3 is allyl, and P 4 is trityl.

8. 2. The method of claim 1, wherein the subunits of the at least one polyamino acid synthon have a D-configuration, an L-configuration, or both a D-configuration and an L-configuration.

9. 2. The method of claim 1, wherein the one carboxylic acid protecting group in step (iv) is removed from the at least one polyamino acid synthon.

10. 2. The method of claim 1, wherein the unprotected carboxylic acid group in step (v) is derived from the at least one polyamino acid synthon.

11. The linear peptide of step (i) is synthesized by (1) coupling at least one amino acid from the plurality of amino acids to the at least one polyamino acid synthon, or (2) coupling at least two amino acids from the plurality of amino acids, or (3) a combination thereof, wherein the coupling occurs via a carbodiimide-mediated reaction or a reaction mediated by a non-carbodiimide coupling agent, and the non-carbodiimide coupling agent is selected from the group consisting of 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 1H-benzotriazolium 1-[bis(dimethylamino)methylene]- 5-chloro-hexafluorophosphate(1-),3-oxide (HCTU), O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), 1-[(1-(cyano-2-ethoxy-2-oxoethylideneaminooxy)-dimethylamino-morpholinomethylene)]methanaminium hexafluorophosphate (COMU), 1-cyano-2-ethoxy-2-oxo 2. The method of claim 1, wherein the hydroxybenzotriazol-1-yloxytris-pyrrolidinophosphonium hexafluorophosphate (HPO) is selected from ethylideneaminooxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyOxim), benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), 7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP), or propanephosphonic anhydride (T3P).

12. 12. The method of claim 11, wherein the carbodiimide is selected from the group of diisopropylcarboxiimide (DIC), dicyclohexylcarbodiimide (DCC), or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).

13. 12. The method of claim 11, wherein the carbodiimide-mediated reaction further comprises an amino acid racemization inhibitor selected from 2-hydroxypyridine-N-oxide (HOPO), 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azo-benzotriazole (HOAt), or 2-cyano-2-(hydroxyimino)acetate.

14. 12. The method of claim 11, wherein the carbodiimide-mediated reaction comprises using a solvent selected from N-methylpyrrolidinone (NMP), dichloromethane (DCM), chloroform, or dimethylformamide (DMF).

15. The global deprotection step (vi) is carried out with ammonium iodide (NH ４ 10. The method of claim 1, comprising adding I) and thioanisole.

16. The method described in claim 1, wherein the modification of the N-terminus of the synthetic peptide is acetylation.

17. Cys １ and Cys ６ Between and Cys ５ and Cys １３ The method of claim 1, wherein the covalent bond between Cth2 and Cys10 is a disulfide bond and the covalent bond between Cth2 and Cys10 is a thioether bond.

18. 3. The method of claim 2, wherein the organic solvent mixture comprises at least one of acetonitrile or methyl tert-butyl ether (MTBE).

19. Formula I: 【Chemistry 42】 1. A method for preparing a synthetic peptide of the formula: (i) A Tyr-Gly peptide containing a protected Tyr amino acid is prepared by the reaction of the peptide with Formula II: 【Chemistry 43】 to a polyamino acid synthon of the C-terminus of the Tyr-Gly peptide is bound to a resin; P 1 , P 2 , P 3 , and P 4 are each a protecting group; During the ceremony, P １ and P ２ are each an amine protecting group, P 1 and P 2 are not the same; P ３ is a carboxylic acid protecting group, P ４ is a thiol protecting group, Coupling the Tyr-Gly peptide to the polyamino acid synthon of formula II yields a polyamino acid synthon of formula III: 【Chemistry 44】 and providing a resin-bound peptide of (ii) the P of formula III ２ The protecting group is removed to give a compound of formula IV having a first free amine group: 【Chemistry 45】 and obtaining a resin-bound peptide of (iii) in the first free amine group, P ２ -alanine is coupled to the resin-bound peptide of formula IV to give the peptide of formula V: 【Chemistry 46】 forming a resin-bound peptide of (iv) the P of formula V ２ Removal of the protecting group to provide a second free amine group, followed by conversion of said second free amine group to P ２ coupling to an amino acid, the amino acid of the P 2 -amino acid is valine; the side chains of the amino acids are protected as needed; and (v) Repeating step (iv) five more times to obtain a compound of formula VI: 【Chemistry 47】 forming a resin-bound peptide of wherein at least one amino acid side chain is protected, the amino acids of the P 2 -amino acids used in each of the five further instances are Asn, Cys, Cys, Leu, and Glu, respectively; (vi) cleaving the resin-bound peptide of formula VI from the resin to form a linear peptide having a C-terminal carboxylic acid group; (vii) coupling the C-terminal carboxylic acid group of the linear peptide to the amine group of a cysteine ​​to form a compound of formula VII 【Chemistry 48】 to obtain a protected peptide of the cysteine ​​comprises a carboxylic acid protecting group; the side chain of said cysteine ​​is optionally protected; In the formula, P ５ is the P ３ a carboxylic acid protecting group different from the protecting group; (viii) the P of formula VII ２ The protecting group is removed to provide a third free amine group, forming the P ３ removing the protecting group to obtain the free carboxylic acid group; (ix) coupling the third free amine group with the free carboxylic acid group to form a compound of formula VIII: 【Chemistry 49】 and obtaining a cyclized peptide of formula (I) (x) globally deprotecting the cyclized peptide to obtain a globally deprotected peptide; (xi) folding the globally deprotected peptide by forming two disulfide bonds to obtain the synthetic peptide of formula I.

20. Acetylation of the free amine group in formula I to form a compound of formula IX: [Transformation 50] 20. The method of claim 19, further comprising obtaining a synthetic peptide of

21. 21. The method of claim 19 or 20, wherein the Glu, Cys, Cys, Asn, Gly and Cys residues of formula VII have side chain protecting groups.

22. The following structural formula: 【Chemistry 51】 (In the formula, P １ and P ２ are each independently hydrogen or an amine protecting group, and if P １ and P ２ are amine protecting groups, the amine protecting groups are not the same, P ３ is hydrogen or a carboxylic acid protecting group, P ４ is hydrogen or a thiol protecting group) or a pharmaceutically acceptable salt thereof.

23. P １ or P ２ is each independently acetyl, fluorenylmethoxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), carboxybenzyl (Cbz), or allyloxycarbonyl (Alloc); P 3 is methyl, ethyl, tert-butyl, allyl, trityl, 2,4-dimethoxybenzyl (Dmb), 9-fluorenylmethyl (Fm), or benzyl (Bn); and P 4 is acetamidomethyl (Acm), tert-butyl (t-Bu), 3-nitro-2-pyridinesulfenyl (NPYS), 2-pyridinesulfenyl (Pys), or trityl (Trt), or a pharmaceutically acceptable salt thereof.

24. P １ 23. The method of claim 19, or the compound of claim 22, or a pharmaceutically acceptable salt thereof, wherein P is acetyl or tert-butyloxycarbonyl (Boc), P 2 is fluorenylmethoxycarbonyl (Fmoc), P 3 is an allyl protecting group, and P 4 is a trityl protecting group or a tert-butyl protecting group.

25. P １ , P ２ , P ３ , or P ４ 23. The compound of claim 22, or a pharmaceutically acceptable salt thereof, wherein at least one of is hydrogen.

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