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 having protected amine groups at its C-terminus and its N-terminus bound 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 at least one polyamino acid synthon. In some embodiments, the synthon has at least one amine group that is acetylated and at least one carboxylic acid protecting group.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 282,851, filed November 24, 2021, the contents of which are incorporated herein by reference in their entirety.

[0002] FIELD OF THEINVENTION 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 XML entitled "223355-519433.xml" (8.11 kilobytes), which was created on November 18, 2022 at 3:45 PM and 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 having a protected amine group at its N-terminus and C-terminus 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 at least one polyamino acid synthon, the synthon having at least one acetylated amine group and at least one carboxylic acid protecting group; and (ii) removing the protecting groups from the carboxylic acid protecting groups of the synthon and the amine group at the N-terminus of the linear peptide to leave an unprotected amine group and an unprotected carboxylic acid protecting group. 1. A method comprising the steps of forming a partially unprotected solid support bound peptide having an acid group, (iii) coupling an unprotected amine group with an unprotected carboxylic acid group to form a cyclized solid support bound peptide, (iv) cleaving the cyclized solid support bound peptide from the solid support to produce a cyclized protected peptide, (v) globally deprotecting the cyclized protected peptide to obtain a globally deprotected peptide, (vi) folding the globally deprotected peptide to form one or more additional crosslinks to obtain a synthetic peptide, and (vii) purifying the synthetic peptide. The synthetic peptide has the amino acid sequence: Ac-Cys1Cth2Glu3Leu4Cys5Cys6Asn7Val8Ala9Cys 10 Tyr 11 Gly 12 Cys 13 (SEQ ID NO:1). The synthetic peptides include the following: Cys1 and Cys6, Cth2 and Cys 10 , and Cys5 and Cys 13 It contains covalent bonds between amino acid residues. [Brief description of the drawings]

[0008] [Figure 1] FIG. 1 shows an exemplary flow diagram for producing a linear synthetic peptide of step (i) of the method described herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] 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 having a C-terminus bound to a solid support and an N-terminus protected amine group using a plurality of amino acids and at least one polyamino acid synthon, said linear peptide having protecting groups on one or more amino acids and / or said at least one polyamino acid synthon; chemically synthesizing said synthon having at least one acetylated amine group and at least one carboxylic acid protecting group; (ii) removing the carboxylic acid protecting group of the synthon and the protecting group from the amine group at the N-terminus of the linear peptide to form a partially unprotected solid support bound peptide having an unprotected amine group and an unprotected carboxylic acid group; (iii) coupling the unprotected amine group with the unprotected carboxylic acid group to form a cyclized solid support-bound peptide; (iv) cleaving the cyclized solid support-bound peptide from the solid support to produce a cyclized protected peptide; (v) globally deprotecting the cyclized protected peptide to obtain a globally deprotected peptide; (vi) folding the globally deprotected peptide to form one or more additional cross-links to obtain the synthetic peptide; (vii) purifying the synthetic peptide, The synthetic peptide has the amino acid sequence: Ac-Cys1Cth2Glu3Leu4Cys5Cys6Asn7Val8Ala9Cys 10 Tyr 11 Gly 12 Cys 13(SEQ ID NO: 1), The synthetic peptide comprises: a) Cys1 and Cys6, b) Cth2 and Cys 10 , and c) Cys5 and Cys 13 It contains covalent bonds between amino acid residues.

[0010] 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]

[0011] 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."

[0012] As used herein, "Hcy" or "Hcys" 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."

[0013] 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.

[0014] 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.

[0015] Synthetic Peptides In some embodiments, the synthetic peptide produced by the methods of the present disclosure is Ac-Cys1Cth2Glu3Leu4Cys5Cys6Asn7Val8Ala9Cys 10 Tyr 11 Gly 12 Cys 13 (SEQ ID NO:1), where "Ac" indicates that the N-terminal amine group is acetylated.

[0016] 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 a homocysteine ​​and a cysteine ​​that share a side chain sulfur atom) that provides an internal sulfide (or thioether) bond with the cystathione (Cth) unit.

[0017] For purposes herein, the two portions of the linear sequence are Cth2 and Cys 10 and 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 the cystathionine (Cth) residue, but the proposed nomenclature makes it easier to interpret when using the three-letter code designation of the residues, where the peptide bond formed by the α-aminocarboxyl group at position 1 of cystathionine is named "Cth" and the peptide bond formed by the α-aminocarboxyl group at position 2 is named "Cys".

[0018] Alternatively, for purposes herein, the two moieties of the building block can be designated [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 corresponds to the cystathionine (Cth) residue, although the proposed nomenclature facilitates explanation when using the three-letter code nomenclature of the residues. Using this alternative nomenclature, SEQ ID NO:1 is represented as follows: Ac-Cys1Hcy2Glu3Leu4Cys5Cys6Asn7Val8Ala9Cys 10 Tyr 11 Gly 12 Cys 13 (Sequence number 1).

[0019] In some embodiments, Cth2-Cys 10 The nomenclature, 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]

[0020] (SEQ ID NO: 1) In some embodiments, Cth2-Cys 10 or any variation thereof, describes cystathionine which forms peptide bonds at positions 2 and 10 of the synthetic peptide, forming a thioether bridge.

[0021] In some embodiments, the synthetic peptide of SEQ ID NO:1 has the formula: [ka] (SEQ ID NO:1) It can be expressed as:

[0022] Methods for Producing Synthetic Peptides The methods described herein begin by (i) chemically synthesizing a linear peptide having protected amine groups at its C-terminus and its N-terminus bound 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 at least one polyamino acid synthon. In some embodiments, the synthon has at least one amine group that is acetylated and at least one carboxylic acid protecting group.

[0023] In some embodiments, the solid support is selected from the group consisting of Wang resin, trityl resin, and Rink resin.

[0024] 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.

[0025] In some embodiments, the polyamino acid synthon has the formula: [ka] (In the formula, P 2 is an amine protecting group, P 3 is a carboxylic acid protecting group, P 4 is a thiol protecting group.

[0026] 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, 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).

[0027] In some embodiments, the amine protecting group P 2 is selected from the group consisting of fluorenylmethyloxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), and carboxybenzyl (Cbz). 2 is a 9-fluorenylmethoxycarbonyl (Fmoc) protecting group.

[0028] In some embodiments, the carboxylic acid protecting group P 3 is selected from the group consisting of methyl, ethyl, tert-butyl, allyl, 2,4-dimethoxybenzyl (Dmb), 9-fluorenylmethyl (Fm), and benzyl (Bn). 3 is an allyl protecting group.

[0029] In some embodiments, P 4 is a trityl protecting group.

[0030] 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]

[0031] 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]

[0032] In some embodiments, the subunits of the polyamino acid synthon have both the D- and L-configurations.

[0033] 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, 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).

[0034] 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.

[0035] In some embodiments, the multiple amino acids and synthons are coupled by carbodiimide mediated reactions or with non-carbodiimide coupling agents: 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 (HBTU), phosphate(1-), 3-oxide (HCTU), O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), 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).

[0036] 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 diisopropylcarboxiimide (DIC), dicyclohexylcarbodiimide (DCC), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). In some embodiments, the carbodiimide is DIC.

[0037] In some embodiments, the carbodiimide mediated reaction further comprises an antioxidant. In some embodiments, the antioxidant is a soluble thiourea or thiol compound. In some embodiments, the antioxidant is 1,3-diisopropyl-2 thiourea (DITU) or dithiothreitol.

[0038] In some embodiments, at least one amino acid is linked by a non-carbodiimide coupling agent. In some embodiments, the cyclization coupling reaction is mediated by a non-carbodiimide coupling agent. In some embodiments, the non-carbodiimide coupling agent used in the cyclization coupling reaction is HATU.

[0039] The linear peptide of step (i) may be referred to in the present application as a "linear 13-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.

[0040] After the linear peptide is formed, (ii) the carboxylic acid protecting group of the synthon and the protecting group from the amine group at the N-terminus of the linear peptide are removed to form a partially unprotected solid support-bound peptide having an unprotected amine group and an unprotected carboxylic acid group. In some embodiments, deprotection is accomplished using Pd(PPh3)4 and 1,3-DMBA in DMF.

[0041] In some embodiments, the partially unprotected solid support bound peptide may be represented by the formula: [ka]

[0042] (SEQ ID NO:3) Following partial deprotection, (iii) the unprotected amine group is coupled with the unprotected carboxylic acid group to form a cyclized solid support-bound peptide.

[0043] In some embodiments, the cyclized solid support bound peptide may be represented by the formula: [ka]

[0044] (SEQ ID NO: 4) The cyclized solid support bound peptide is (iv) cleaved from the solid support to generate a cyclized protected peptide.

[0045] In some embodiments, the linear peptide is cleaved from the resin by dilute acid treatment. In some embodiments, the dilute acid treatment preserves the side chain protecting groups and the protecting groups of the polyamino acid synthons. In some embodiments, the dilute acid treatment is a weak acid solution, such as trifluoroacetic acid (TFA). In some embodiments, the dilute acid solution is a trifluoroacetic acid (TFA) solution. In some embodiments, the dilute acid solution is a 1% trifluoroacetic acid (TFA) in dichloromethane (DCM) solution.

[0046] Once the cyclized protected peptide is cleaved from the resin, the peptide is (v) globally deprotected to obtain a globally deprotected peptide. In some embodiments, the global deprotection step (v) comprises the addition of a cocktail comprising at least ammonium iodide (NH4I). In some embodiments, the global deprotection step (v) comprises the addition of a cocktail comprising at least ammonium iodide (NH4I) and triisopropylsilane.

[0047] Once the cyclized peptide is globally deprotected, the peptide (vi) folds back to form one or more additional bridges, forming Ac-Cys1Cth2Glu3Leu4Cys5Cys6Asn7Val8Ala9Cys 10 Tyr 11 Gly 12 Cys13 A synthetic peptide of (SEQ ID NO: 1) is obtained.

[0048] As used herein, "folding" and "oxidation" may refer to the same process where folding is accomplished via oxidation of the cysteine ​​residues of SEQ ID NO:1. In some embodiments, the folding step (vi) is accomplished by iodine-mediated oxidation or alkali-mediated oxidation. In some embodiments, the alkali-mediated oxidation is dimethylsulfoxide (DMSO)-mediated oxidation or N-methyl-2-pyrrolidone (NMP)-mediated oxidation.

[0049] In some embodiments, the synthetic peptide is provided by isolating 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.

[0050] Following folding of the globally deprotected peptide, the synthetic peptide of SEQ ID NO:1 is purified.

[0051] In some embodiments, the synthetic peptide of SEQ ID NO:1 has the formula: [ka] (SEQ ID NO:1) It can be expressed as:

[0052] Also described herein is a compound of formula I: [ka] A method for preparing a synthetic peptide of the formula: (i) preparing a C-terminal resin-bound Tyr-Gly-Cys peptide having protected amino acid side chains, according to formula II: [ka] (In the formula, P 2 is an amine protecting group, P 3 is a carboxylic acid protecting group, P 4 is a thiol protecting group) to a polyamino acid synthon of formula III: [ka] forming a resin-bound peptide of (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 a 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 five 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) Said P 2 Protecting groups and the P 3removing the protecting groups to provide a free amine group and a free carboxylic acid group; (vii) coupling the free amine group with the free carboxylic acid group to form a compound of formula VII: [ka] and obtaining a cyclized peptide of the formula: (viii) cleaving the peptide of formula VII from the resin to obtain a cyclized peptide; (ix) globally deprotecting the cyclized peptide to obtain a globally deprotected peptide; (x) folding the globally deprotected peptide by forming two disulfide bonds to obtain the synthetic peptide of formula I.

[0053] In some embodiments, the Glu, Cys, Cys, Asn, Gly and Cys residues of formula VI 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).

[0054] In some embodiments, P 2 is a protecting group selected from the group consisting of fluorenylmethoxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), carboxybenzyl (Cbz), and allyloxycarbonyl (Alloc). 2 is a fluorenylmethoxycarbonyl (Fmoc) protecting group.

[0055] 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.

[0056] In some embodiments, P 4 is a protecting group selected from the group consisting of acetamidomethyl (Acm), tert-butyl (t-But), 3-nitro-2-pyridinesulfenyl (NPYS), 2-pyridinesulfenyl (Pyr), and trityl (Trt). 4 is a trityl protecting group. EXAMPLES

[0057] 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.

[0058] Abbreviation AA: Amino acid AAA: Amino acid analysis Ac: Acetyl AcOH: Acetic acid All or Allyl: 2-Propenyl API: Active Pharmaceutical Ingredient BB: Has the following structure (Fmoc-Cys 10* [Ac-Cys(Trt)-Hcys(S * )-OAll]-OH): [ka] C18: Silica gel C18 DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene DIC: Diisopropylcarbodiimide DIPEA: Diisopropylethylamine DITU: 1,3-Diisopropyl-2-thiourea 1,3-DMBA: 1,3-dimethylbarbituric acid DMF: N,N-dimethylformamide DMSO: Dimethyl sulfoxide DTT: dithiothreitol EtOH: Ethanol eq: equivalent Fmoc: 9-fluorenylmethoxycarbonyl GC: Gas Chromatography GSH / GSSG: Glutathione (red / ox) HATU: 1H-1,2,3-triazolo[4,5-b]pyridinium, 1-[bis(dimethylamino)methylene]-, 3-oxide, hexafluorophosphate(1-)(1:1) HDPE: High density polyethylene HPLC: High performance liquid chromatography IPA: Isopropanol LC: Liquid chromatography MeCN: Acetonitrile MeOH: Methanol MTBE: Methyl tert-butyl ether MS: mass spectrometry Mw: molecular weight NH4OAc: Ammonium acetate NMM: N-methylmorpholine NMP: N-methylpyrrolidone NMR: nuclear magnetic resonance Oxyma: Ethyl (hydroxyimino)cyanoacetate PPh3: Triphenylphosphine SEC: Size Exclusion Chromatography SPPS: Solid Phase Peptide Synthesis tBu: tert-butyl TFA: Trifluoroacetic acid Thz: thiazolidinedione Thz(Me)2: 2,2-dimethylthiazolidine TIS: Triisopropylsilane Trt: triphenylmethyl Trt-H: Triphenylmethane UV: Ultraviolet light

[0059] Example 1 Process for producing synthetic peptide of SEQ ID NO:1 The peptide of SEQ ID NO:1 or a pharma- ceutically acceptable salt thereof was manufactured in accordance with Good Manufacturing Practice (GMP) regulations.

[0060] material Fmoc-D-Cys(Trt)-OH (>97.0%, HPLC) and TFA (>99.0%, HPLC) for HPLC analysis were purchased from Sigma-Aldrich. Fmoc-Thz-OH (99.6%, HPLC) and Fmoc-Thz(Me)2-OH (>99.3%, HPLC; 99.7%ee) were obtained from PepTech. BB (>98.5%, HPLC) was custom synthesized. BB, L-enantiomer identity was confirmed by NMR and chiral analysis. DITU from Molekula, DMSO(pa) from Riedel-de Hahn, and O-methylhydroxylamine hydrochloride from Merck were used. GSH and GSSG were obtained from Sigma-Aldrich. All other reagents, amino acid derivatives, 2-CTC resin and solvents were obtained from storage. All materials were used as received. Purified water was used.

[0061] device synthesis equipment

[0062] Special stoppered syringes equipped with filters were used for small-scale SPPS and cleavage / deprotection. The syringes with their contents were shaken automatically at ambient temperature (20-25 °C).

[0063] Larger scale SPPS utilized a jacketed reactor equipped with an overhead stirrer. Temperature was controlled in the jacketed reactor with a cryostat (Julabo).

[0064] Cleavage / deprotection starting from >5 g peptide resin was carried out in glassware or sealed HDPE bottles.

[0065] Iodine-mediated oxidation (disulfide formation) was carried out using a syringe pump and appropriate TEFLON® tubing.

[0066] Otherwise, standard laboratory equipment / instruments were used for the synthetic work.

[0067] Occasionally, a Beckman centrifuge (3750-4000 rpm, 10 min) was used for small-scale precipitation of the cleaved / deprotected peptide.

[0068] analytical equipment

[0069] HPLC was performed at 30° C. on an Agilent system equipped with an XSelect Peptide 130A 150×4.6 mm 2.5 μ column. Mobile phase A: 0.1% TFA (aqueous); Mobile phase B: 0.1% TFA (MeCN).

[0070] LC / MS used the same column on a Thermo Scientific Vanquish Horizon UHPLC interfaced to an ESI Thermo Q-Exactive MS Spectrometer. Chromeleon software was used for HPLC / MS evaluation.

[0071] Chiral AAA was produced at CAT GmbH & Co. Chromatographie and Analysentechnik KG (Tübingen, Germany).

[0072] NMR analyses were performed by RED GLEAD DISCOVERY AB (Medicon Village, Lund 223 81, Sweden).

[0073] Peptide synthesis SPPS

[0074] FIG. 1 shows a flow diagram of the SPPS synthesis of a solid-bound 13-mer.

[0075] Standard protocols were used. After drying, all peptide resins were stored in the freezer.

[0076] LH-Cys-2-CT resin or DH-Cys-2-CT resin

[0077] L-Cys or D-Cys was coupled to the 2-CTC resin for approximately 5 h at 20-25 °C in an SPPS reactor equipped with a glass filter, and the remaining binding sites on the resin were capped with methanol. After draining / washing, the Fmoc groups were removed by treatment with 20% (v / v) piperidine in DMF (2 × 10 min). The resin was washed with DMF until a negative chloranil test indicating removal of piperidine, then with isopropanol, and finally dried in vacuum at 20-25 °C for 1-2 days. L- H-Cys-2-CT resin; 102.0 g, 0.42 mmol / g (triphenylmethane) D- H-Cys-2-CT resin; 14.5 g, 0.40 mmol / g (triphenylmethane)

[0078] Fmoc(7-13)-2-CT resin

[0079] L-Cys 13 In this version a jacketed reactor was used. D- Cys 13 In the case of 20℃(T ジャケット ) and the reaction was carried out at 20 to 25°C.

[0080] Coupling was carried out in either DMF or NMP. 13 Relative to the loading, about 2.0 equivalents of AA derivative were used, except for BB (1.5 equivalents). About 2.2 equivalents of Oxyma / DIC and about 0.2 equivalents of DITU were used. Glycine was preactivated for 1 hour. Additional DIC (about 2.2 equivalents) was generally added after some time. Total coupling times were generally 2-3 hours, and about 5 hours for BB.

[0081] Glycine was preactivated to accelerate Gly coupling and thus avoid unintended detachment of Cys from the acid-sensitive 2-CT resin by slightly acidic Oxyma or amino acids, potentially resulting in endo-Cys and des-Cys.

[0082] After each coupling, the reactor was emptied and the resin was washed with DMF. The peptide resin was stored in the reactor (5-10° C.) when the synthesis was paused overnight. The synthesis was continued after increasing the operating temperature.

[0083] The Fmoc group was removed by treatment with 20% (v / v) piperidine in DMF (2×10 min) and the resin was washed extensively until a chloranil test was negative, indicating complete removal of piperidine.

[0084] Once the sequence was complete, it was washed with DMF and isopropanol, and then the resin was dried under vacuum at 20-25°C for 1-2 days.

[0085] To promote cyclization through the cis-inducing conformation, Cys (5または6) One of the residues was incorporated as a pseudoproline. The appropriate derivatives of choice could be Fmoc-L-Thz-OH (CAS number [133054-21-4]) and Fmoc-L-Thz(Me2)-OH (CAS number [873842-06-9]). Thz could replace Cys(Trt) at the 6 or 5 position, or both positions. Thus, four different Fmoc(3-13)-2-CT resin peptides were synthesized as shown below: [ka] Yield based on theoretical product weight.

[0086] Ring closure / lactamization

[0087] Fmoc(3-13)-2-CT resin was swollen in a syringe with DMF. After draining, Fmoc was removed with 20% piperidine (>10 min +>20 min). The resin was washed extensively until the chloranil test was negative. The allyl ester of Hcys was converted to the acid overnight with approx. 10 mol% Pd(PPh3)4 in DMF and approx. 10 equiv. of 1,3-DMBA. The resin was drained, washed with DMF, and ring-closed with approx. 2 equiv. of HATU and 2.7 equiv. of NMM. Kaiser test indicated complete reaction within 4-5 h. Final washing and drying were performed as above.

[0088] Cleavage / Deprotection

[0089] Analytical scale cleavage / deprotection was carried out in a syringe as described below: The TFA solution was filtered into ice-cold (-18°C) diethyl ether, the precipitate was centrifuged, the supernatant was decanted, the residue was triturated with diethyl ether, and the solid was centrifuged again.

[0090] Preparative scale (Ac-Cys(H)-Hcys(S1-Glu-Leu-Cys(H)-Cys(H)-Asn-Val-Ala-Cys * -Tyr-Gly-Cys(H)-OH): Cyclized peptide resin Cys 5および6(Trt) (5.41 g) was mixed with 1.8 g DTT, 1.7 g NHI, 4.5 mL TIS, 1.8 mL water and 50 mL TFA. The mixture was stirred for 3 h. The resin was filtered off and washed with 2 × 15 mL TFA. The combined filtrate was cooled (10 °C) and ice-cold (-18 °C) diethyl ether (360 mL) was added portionwise with stirring for 10-15 min (T. 23 °C). Stirring was continued at 0 °C for about 5 min. The solid was filtered (16-40 μm) and washed with diethyl ether (2 × 100 mL). It was then dried under vacuum at 20-25 °C for 2 days. This gave 1.80 g of crude cyclic peptide (free-SH).

[0091] Oxidation (formation of SS crosslinks)

[0092] These experiments were performed with the crude cyclic peptide (free-SH) obtained from Fmoc-Glu(OtBu)-Leu-Cys(Trt)-Cys(Trt)-Asn(Trt)-Val-Ala-BB-Tyr(tBu)-Gly-Cys(Trt)-2CTC-resin. All oxidations were performed at 20–25 °C.

[0093] Iodine Oxidation

[0094] A solution of crude cyclized peptide in DMSO (10 g / L) was made. The solution was diluted 10-fold with 20% MeCN (aq) and became slightly cloudy (pH approx. 4). To this solution, iodine in MeCN (1% w / v) was added dropwise over approximately 1 h until a yellow / brown color persisted. The iodine was quenched with aqueous ascorbic acid (0.5 M). The pH was raised to 7-7.5 with 3.5% NH3 (aq). GSH / GSSG was added at different time points (1-3.5 mM).

[0095] DMSO oxidation

[0096] Clear solutions were made with DMSO. These were diluted to 30% DMSO in water (pH approx. 4) to give concentrations of 1-3.3 g / L. This slightly cloudy mixture was stirred at 60 °C or the pH was adjusted to 7.5-8 with 3.5% NH3 (aq). At the latter pH the solution was completely clear again. GSH / GSSG was added if necessary.

[0097] NMP oxidation

[0098] A clear solution of 0.5 g crude / ml NMP was made.

[0099] Results and Discussion H-Cys(Trt)-2-CT resin and Fmoc(7-13)-2-CT resin

[0100] A Cys loading of 0.5 mmol / g resin was targeted. The consumption of Fmoc-Cys(Trt)-OH was followed by HPLC of the reaction solution. At the stage of incorporation of 54 mmol (approximately 5 h), corresponding to approximately 0.6 mmol / g, the reaction was stopped by washing. The loading was found to be 0.42 mmol / g.

[0101] From this Cys resin, we made the intermediate Fmoc (7-13) resin, which is common to all the proposed (3-13) candidates for ring closure of the resin.

[0102] Based on the Fmoc loading (0.199 mmol / g), the yield of Fmoc(7-13)-2-CT resin (61.9 g) was 81.6%. The purity of the cleaved Fmoc(7-13)OH without side chain protection was about 85% (as measured by HPLC).

[0103] Some peptides may be cleaved by prolonged action of slightly acidic Oxyma because the ester bond to 2-CT is acid sensitive. Treatment of Fmoc(7-13)-2-CT resin with approximately 3 equivalents of Oxyma in DMF for 21 h (20-25 °C) did not result in loss of peptide.

[0104] DMF was used throughout, but NMP was also tested when performing the couplings, the latter solvent did not provide any improvement.

[0105] DH-Cys(Trt)-2-CT resin (0.40 mmol / g) and [DH-Cys 13 (Trt)]Fmoc(7-13)-2-CT resin (0.175 mmol / g) was added to L-Cys 13 It was made in the same way as version 1.

[0106] Fmoc(3-13)-2-CT resin

[0107] Syringe synthesis of the Fmoc(3-13)-2-CT peptides was carried out from 7.5 g of Fmoc(7-13) resin each. Coupling was carried out using Oxyma / DIC for 3-5 hours, with additional DIC added after approximately 2 hours. [ka] Yield based on theoretical product weight.

[0108] After final washing / drying, a small sample was cleaved / deprotected and subjected to further analysis. Best results were observed using Cys(Trt) at both positions 5 and 6. Coupling to already incorporated Thz was slower (4-5 h). The Thz(Me)2 variant lacks the Fmoc-Gly-Leu.

[0109] To overcome the reluctance of the H-Thz(Me2)5-Cys(Trt)-Asn(Trt)-Val-Ala-BB-Tyr(tBu)-Gly-Cys(Trt)-2-CT resin to continue coupling, it was again swollen in DMF and treated with HATU / NMM and Fmoc-Leu-OH at 30° C. overnight. An activated Fmoc-Leu-OAt was formed, which is believed to be more reactive towards hindered amines. Unfortunately, this was unsuccessful.

[0110] The estimated yield of the baseline Fmoc-Glu(OtBu)-Leu-Cys(Trt)-Cys(Trt)-Asn(Trt)-Val-Ala-BB-Tyr(tBu)-Gly-Cys(Trt)-2CT resin was 48% (based on Fmoc loading and purity of the (7-13) and (3-13) intermediates) compared to the H-Cys(Trt)-2-CT resin.

[0111] On-resin ring closure (lactamization)

[0112] Cyclization was achieved by first removing Fmoc from the (3-13) resin, followed by Pd-catalyzed deallylation of Hcys and HATU-assisted cyclization (coupling of -Hcy-OH to H-Glu-) for 4-5 h. The reaction steps were carried out using the following resins: [ka]

[0113] Among the all-L analogs, the baseline resin gave the purest lactam, but the Kaiser test did not show any significant difference in the cyclization rate. Recall that the purity of the linear Thz resin was somewhat lower compared to the baseline resin due to incomplete coupling.

[0114] The estimated yield of the baseline resin was 32% (relative to H-Cys(Trt)-2-CT resin) based on the Fmoc / Trt loading and purity of the (7-13), (3-13) and (1-13) intermediates.

[0115] From the baseline resin (5.41 g), cyclization followed by cleavage / deprotection / precipitation afforded the lactam shown below in a quantity of 1.80 g. [ka]

[0116] The measured content of D-Cys in baseline lactams was 0.01–0.02%. 13This indicates that epimerization of is at least not very significant.

[0117] Oxidation (formation of SS crosslinks)

[0118] Oxidation is the final step.

[0119] The baseline Ac(1-13)-OH cyclization product is sufficiently soluble in DMF for HPLC analysis, but making a 10 g / L solution appears difficult. It appears very insoluble in MeCN, 20-80% MeCN (aqueous with or without AcOH), neat AcOH, 25% AcOH (aqueous), water, EtOH, and MeOH. It is very soluble in DMSO, and the solution is stable when kept at 5-10°C and even 25°C for several days (HPLC). Diluting 10-fold with 20% MeCN (aqueous) caused the resulting mixture to become somewhat hazens, but there was no peptide loss upon filtration (HPLC). It is readily soluble in NMP (10 g / L), but reacts quickly.

[0120] Alkaline DMSO oxidation was the simplest method. Decreasing the crude concentration from 3 g / L to 1 g / L gave virtually identical results, with no increase in purity observed compared to the 3 g / L experiment. Increasing the folding time from 1 to 2 days did not change the HPLC profile, regardless of crude lactam concentration.

[0121] It is not clear whether GSH / GSSG actually affects folding upon iodine oxidation or whether increasing pH alone alters the HPLC profile; at least in the case of alkaline DMSO oxidation, GSH-GSSG played no discernible role.

[0122] NMP as a solvent was not useful because the peptide reacted very rapidly to an undefined mixture. The high reactivity is most likely due to NMP(5-OOH) that forms in NMP upon aging under oxidizing conditions (air).

[0123] conclusion The strategy shown in FIG. 1, for example, a synthetic peptide of SEQ ID NO:1 can be synthesized using a standard SPPS protocol for assembling amino acids into a desired sequence, followed by cyclization via HATU-assisted amide bond formation between Hcys and Glu to give the macrocyclic protected peptide on resin shown below. [ka]

[0124] The estimated yield of this cyclic peptide on resin based on H-Cys(Trt)-2-CT resin was 32%, which corresponds to a cyclization yield of 67%.

[0125] Analogs with Thz at the 5 or 6 positions or both provided no advantage. Coupling to an already incorporated Thz requires more time to complete. Substitution with these Cys analogs would further require an additional process step to remove the methylene bridge with aqueous MeONH2 to remove formaldehyde prior to disulfide bridge construction. They were not deprotected by TFA cocktail.

[0126] Another Cys derivative, Fmoc-Thz(Me)2-OH, was also investigated, which was easily converted to Cys by TFA deprotection but did not allow further coupling when incorporated into the peptide sequence.

[0127] Therefore, the cyclized baseline resin shown above is the best choice. Without Thz in the intermediate peptide sequence, analytical methods to detect possible residual formaldehyde would be unnecessary.

[0128] Upon ring closure, cleavage / deprotection / precipitation (1.80 g from 5.41 g baseline resin) gave the following: [ka] This gives the lactam shown in Figure 1, which may also be represented by the formula [ka]

[0129] Oxidation, e.g., formation of a disulfide from the lactam, was performed either with iodine or in a DMSO-containing solution, resulting in the formation of correctly folded products (the best choice so far has been alkaline DMSO oxidation (HPLC / SEC)).

[0130] 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.

[0131] 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 an N-terminal protected amine and C-terminally 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 comprising at least one acetylated amine group and at least one carboxylic acid protecting group; (ii) removing the carboxylic acid protecting group of the at least one polyamino acid synthon and the protecting group of the protected amine at the N-terminus of the linear peptide to form a partially unprotected solid support-bound peptide having an unprotected amine group and an unprotected carboxylic acid group; (iii) coupling the unprotected amine group with the unprotected carboxylic acid group to form a cyclized solid support-bound peptide; (iv) cleaving the cyclized solid support-bound peptide from the solid support to produce a cyclized protected peptide; (v) globally deprotecting the cyclized protected peptide to obtain a globally deprotected peptide; (vi) folding the globally deprotected peptide to form one or more additional cross-links to obtain the synthetic peptide; The synthetic peptide has the amino acid sequence: Ac-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), The synthetic peptide comprises the following amino acid residues: 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 purifying the synthetic peptide from solution, optionally by lyophilization.

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 27】 (In the formula, P ２ is an amine protecting group, P ３ is a carboxylic acid protecting group, P ４ is a thiol protecting group.

6. 6. The method of claim 5, 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 (Pyr).

7. P ３ 6. The method of claim 5, wherein is an allyl protecting group and P4 is a trityl protecting group.

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. The linear peptide of step (i) is synthesized by (1) coupling at least one amino acid from the plurality of amino acids with 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 is performed by a carbodiimide-mediated reaction, or by a method using 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-benzotriazol-1-yl-2-methyl-3-pyridinium ...

10. The method of claim 1, wherein the coupling occurs via a reaction mediated by a non-carbodiimide coupling agent selected from triazolium 1-[bis(dimethyl-amino)methylene]-5-chloro-hexafluorophosphate(1-),3-oxide (HCTU), O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), 7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP), or propanephosphonic anhydride (T3P).

10. 10. The method of claim 9, wherein the carbodiimide is selected from the group of diisopropylcarboxiimide (DIC), dicyclohexylcarbodiimide (DCC) or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).

11. 10. The method of claim 9, wherein the carbodiimide-mediated reaction further comprises an antioxidant.

12. 12. The method of claim 11, wherein the antioxidant is 1,3-diisopropyl-2 thiourea (DITU) or dithiothreitol.

13. 10. The method of claim 1, wherein the cyclization coupling reaction is mediated by a non-carbodiimide coupling agent.

14. 14. The method of claim 13, wherein the non-carbodiimide coupling agent is HATU.

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

16. 10. The method of claim 1, wherein the folding step (vi) is accomplished by iodine-mediated oxidation or alkali-mediated oxidation.

17. 17. The method of claim 16, wherein the alkali-mediated oxidation is dimethyl sulfoxide (DMSO)-mediated oxidation or N-methyl-2-pyrrolidone (NMP)-mediated oxidation.

18. 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.

19. Formula I: 【Chemistry 28】 1. A method for preparing a synthetic peptide of the formula: (i) Binding a Tyr-Gly-Cys peptide containing protected amino acid side chains to a compound of formula II: 【Chemistry 29】 to a polyamino acid synthon of the C-terminus of the Tyr-Gly-Cys peptide is bound to a resin; P 2 , P 3 , and P 4 are each a protecting group; During the ceremony, P ２ is an amine protecting group, P ３ is a carboxylic acid protecting group, P ４ is a thiol protecting group. Coupling the Tyr-Gly-Cys peptide to the polyamino acid synthon of formula II yields a polyamino acid synthon of formula III: 【Transformation 30】 and (ii) P of formula III ２ The protecting group is removed to give a compound of formula IV having a first free amine group: 【Chemistry 31】 and obtaining a resin-bound peptide of (iii) in the first free amine group, P ２ -alanine to the resin-bound peptide of formula IV to give the compound of formula V: 【Chemistry 32】 forming a resin-bound peptide of (iv) 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 P2 amino acid is valine; The P ２ the side chains of the amino acids may be protected; and (v) Repeating step (iv) five more times to obtain a compound of formula VI: 【Transformation 33】 wherein the P2 amino acids used for each of the five additional times are Asn, Cys, Cys, Leu, and Glu, respectively; at least one amino acid side chain is protected; and (vi) Said P ２ Protecting groups and the P ３ removing the protecting groups to provide a third free amine group and a free carboxylic acid group; (vii) coupling the third free amine group with the free carboxylic acid group to form a compound of formula VII: 【Transformation 34】 and obtaining a resin-bound cyclized peptide of formula (I) (viii) cleaving the resin-bound cyclized peptide of formula VII from the resin to obtain a cyclized peptide; (ix) globally deprotecting the cyclized peptide to obtain a globally deprotected peptide; (x) folding the globally deprotected peptide by forming two disulfide bonds to obtain the synthetic peptide of formula I.

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

21. P ２ The method of claim 19 or 20, wherein P is 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-But), 3-nitro-2-pyridinesulfenyl (NPYS), 2-pyridinesulfenyl (Pyr), or trityl (Trt).

Images