CN118591551A - Synthetic methods for producing modified GCC receptor agonists - Google Patents

Abstract

The present invention relates to a method for producing a synthetic peptide of SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof.

Description

Synthetic methods for producing modified GCC receptor agonists

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of U.S. Provisional Application No. 63/282,842, filed on November 24, 2021, and priority to and the benefit of U.S. Provisional Application No. 63/323,552, filed on March 25, 2022, the contents of which are incorporated herein by reference in their entirety.

Technical Field

The present invention relates to a method for producing a synthetic peptide of SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof.

Sequence Listing

This application incorporates by reference in its entirety the sequence listing entitled "223355-519432.xml" (7.73 kilobytes), which was created on November 21, 2022 at 9:49 AM and is submitted electronically herewith.

Background Art

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic condition involving bladder pain that is often accompanied by urinary urgency, frequency, and/or nocturia. IC/BPS is often misdiagnosed as a urinary tract infection, and antibiotics are often 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 etiology of IC/BPS, and it is unclear whether IC/BPS is a primary condition or a secondary result of another condition [Hanno et al. 2015, 193; 1545-1553]. There is no diagnostic test for IC/BPS, and the diagnosis is usually based on urgent and frequent urinary symptoms accompanied by bladder-related pain. The diagnosis is usually reserved until other diseases that may cause these symptoms are ruled out.

There are few approved therapies available for IC/BPS. Patients typically begin treatment with non-pharmacological treatments (general relaxation, stress management, behavioral modification, and physical therapy techniques). Because the therapies available for IC/BPS are minimally effective, many patients utilize off-label therapies, including intravesical instillations (i.e., drug cocktails delivered directly to the bladder via a catheter), to relieve their symptoms. There is a need for more effective, well-tolerated treatments for IC/BPS.

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, there is a need for an efficient synthesis and purification method.

Summary of the invention

The present invention relates to a method for producing a synthetic peptide or a pharmaceutically acceptable salt thereof. The method has the following steps: (i) using a plurality of amino acids and at least one polyamino acid synthon to chemically synthesize a linear peptide whose C-terminus is bound to a solid support, the linear peptide having a protecting group in one or more amino acids and/or polyamino acid synthons; wherein at least one amine group of the polyamino acid synthon has a protecting group different from the N-terminus of the linear peptide; (ii) cleaving the linear peptide from the solid support to generate a protected peptide; (iii) coupling an amino acid to the C-terminus of the protected peptide; (iv) removing an amine protecting group and a 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 the unprotected amine to the unprotected carboxylic acid group to form a cyclized peptide; (vi) fully deprotecting the cyclized peptide to obtain a fully deprotected peptide; (vii) folding the fully 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 comprises 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). The synthetic peptide contains covalent bonds between the following amino acid residues of the synthetic peptide: Cys ₁ and Cys ₆ , Cth ₂ and Cys ₁₀ , and Cys ₅ and Cys ₁₃ .

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

Also disclosed herein is a compound or a pharmaceutically acceptable salt thereof, which is represented by the following structural formula:

wherein ^P1 and ^P2 are hydrogen or an amine protecting group, provided that when ^P1 and ^P2 are both amine protecting groups they are not the same amine protecting group; ^P3 is hydrogen or a carboxylic acid protecting group; and ^P4 is hydrogen or a thiol protecting group.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an exemplary flow chart for making the synthetic peptide of SEQ ID NO:1.

DETAILED DESCRIPTION

Methods for producing synthetic peptides or pharmaceutically acceptable salts thereof are described herein. The methods described herein include:

(i) using a plurality of amino acids and at least one polyamino acid synthon to chemically synthesize a linear peptide whose C-terminus is bound to a solid support, the linear peptide having a protecting group in one or more amino acids and/or polyamino acid synthons;

wherein at least one amine group of the polyamino acid synthon has a protecting group different from that of the N-terminus of the linear peptide;

(ii) cleaving the linear peptide from the solid support to generate a protected peptide;

(iii) coupling an amino acid to the C-terminus of the protected peptide; wherein the amino acid has an unprotected amine group, a protected carboxylic acid group and an optionally protected amino acid side chain.

(iv) removing an amine protecting group and a carboxylic acid protecting group of the protected peptide to form a partially unprotected peptide having an unprotected amine group and an unprotected carboxylic acid group;

(v) coupling an unprotected amine to an unprotected carboxylic acid group to form a cyclized peptide;

(vi) fully deprotecting the cyclized peptide to obtain a fully deprotected peptide;

(vii) folding the fully deprotected peptide to form one or more additional cross-links to obtain the 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 comprises 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 IDNO: 1); and

Wherein the synthetic peptide contains covalent bonds between the following amino acid residues of the synthetic peptide: a) Cys ₁ and Cys ₆ , b) Cys ₂ and Cys ₁₀ , and c) Cys ₅ and Cys ₁₃ .

definition

As used herein, "Cth" means cystathionine, which has two α-aminocarboxyl groups, designated as "1" and "2" in Scheme 1, which can form a peptide bond.

However, to facilitate the use of the 3-letter amino acid code for describing peptide sequences, when a cyclic peptide sequence is generated by forming peptide bonds with each α-aminocarboxyl group (designated "1" and "2") at non-consecutive positions in the peptide sequence, thereby generating a cyclic thioether bridge, the peptide bond formed by the α-aminocarboxyl group at position 1 is designated "Cth", and the peptide bond formed by the α-aminocarboxyl group at position 2 is designated "Cys". See the section entitled "Synthetic Peptides" for further details.

As used herein, "Hcy" means homocysteine as shown in Scheme 1. As can be seen from Scheme 1, cystathionine can be viewed as a combination of homocysteine and cysteine, wherein their side chains share a sulfur atom. Therefore, an alternative method of naming a cyclic peptide sequence generated by forming peptide bonds with each α-aminocarboxyl group of cystathionine at non-contiguous positions in the peptide sequence is to name the peptide linkage formed by the α-aminocarboxyl group at position 1 as "Hcy" and the peptide linkage formed by the α-aminocarboxyl group at position 2 as "Cys".

As used herein, unless otherwise indicated, "pharmaceutically acceptable" means biologically and pharmacologically compatible for in vivo use in animals or humans, and preferably means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly for use in humans.

As used herein, unless otherwise indicated, the terms "about" and "approximately" mean within an acceptable error range for a particular value determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to the practice in the art, "about" can mean within 1 or more than 1 standard deviation. Alternatively, "about" with respect to a composition can mean a range of plus or minus up to 20%, preferably a range of up to 10%. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude of a value, preferably within 5 times of a value, and more preferably within 2 times of a value. Specific values are described in the present application and claims, and unless otherwise indicated, the term "about" means within an acceptable error range for a particular value.

Synthetic peptides

In some embodiments, the synthetic peptide produced by the methods of the present disclosure can be represented linearly as 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 of SEQ ID NO: 1 contains four cysteine residues forming two disulfide bonds and a cystathionine (Cth) unit (which combines homocysteine and cysteine sharing a side chain sulfur atom) providing an internal sulfide (or thioether) bond with defined linkages (Cys ₁ -Cys ₆ , Cys ₅ -Cys ₁₃ , Cth ₂ -Cys ₁₀ ).

For the purposes of this specification, the two parts of the linear sequence are named Cth ₂ and Cys ₁₀ , in which a thioether bond connects the sulfur of the homocysteine (Hcy) side chain to the carbon of the des-SH cysteine side chain: this diamino acid corresponds to a cystathionine (Cth) residue, but the proposed nomenclature aids the description when the three-letter code for the residues is used, in which the peptide linkage formed by the α-aminocarboxyl group at position 1 is named "Cth" and the peptide linkage formed by the α-aminocarboxyl group at position 2 is named "Cys", see Scheme 1 above.

Alternatively, and for the purposes of this specification, the two parts of the building block can be named [Hcy] and [Cys], respectively, where the sulfur of the side chain of homocysteine (Hcy) is shared with the side chain of cysteine (Cys) to form a thioether bridge: this diamino acid corresponds to a cystathionine (Cth) residue, but the proposed nomenclature facilitates description when the 3-letter code for the residue is used. Using this alternative nomenclature, SEQ ID NO 1 can be represented as follows: Cys ₁ Hcy 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).

In some embodiments, the nomenclature of Cth ₂ -Cys ₁₀ or any variation thereof is intended to describe the connection between the side chains of two non-contiguous amino acids in SEQ ID NO 1, which form a thioether bridge as shown below:

In some embodiments, the nomenclature _Cth2 - _Cys10 , or any variation thereof, describes cystathionine that forms a peptide bond at positions 2 and 10 of a synthetic peptide and forms a thioether bridge.

In some embodiments, the synthetic peptide of SEQ ID NO: 1 can be represented by the following formula:

Methods for producing synthetic peptides

The methods described herein begin with: (i) chemically synthesizing a linear peptide having its C-terminus bound to a solid support using a plurality of amino acids and at least one polyamino acid synthon, wherein the linear peptide has a protecting group in one or more amino acids and/or polyamino acid synthons. In some embodiments, at least one amine group of the polyamino acid synthon has a protecting group different from that of the N-terminus of the linear peptide.

In some embodiments, the solid support is selected from Wang resin, Trityl resin, and Rink resin.

In some embodiments, the solid phase carrier has the following load: about 0.10mmol/g, about 0.20mmol/g, about 0.30mmol/g, about 0.40mmol/g, about 0.50mmol/g, about 0.60mmol/g, about 0.70mmol/g, about 0.80mmol/g, about 0.90mmol/g or about 1.00mmol/g. In some embodiments, the solid phase has a load of about 0.70mmol/g. In some embodiments, the solid phase has a load of about 0.90mmol/g.

In some embodiments, the polyamino acid synthon is a compound represented by the formula:

wherein ^P1 and ^P2 are hydrogen or an amine protecting group, provided that when ^P1 and ^P2 are both amine protecting groups they are not the same amine protecting group; ^P3 is hydrogen or a carboxylic acid protecting group; and ^P4 is hydrogen or a thiol protecting group.

In a preferred embodiment, ^P1 and ^P2 are different amine protecting groups; ^P3 is a carboxylic acid protecting group; and ^P4 is a thiol protecting group.

In some embodiments, the protecting group is selected from fluorenylmethoxycarbonyl (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-pyridylsulfenyl (NPYS) and 2-pyridyl-sulfenyl (Pyr).

In some embodiments, amine protecting groups ^P1 and ^P2 are each selected from fluorenylmethoxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc) and carboxybenzyl (Cbz). In some embodiments, ^P1 or ^P2 is a tert-butyloxycarbonyl (Boc) protecting group. In some embodiments, ^P1 or ^P2 is a 9-fluorenylmethoxycarbonyl (Fmoc) protecting group. In some embodiments, ^P1 is a tert-butyloxycarbonyl (Boc) protecting group, and ^P2 is a 9-fluorenylmethoxycarbonyl (Fmoc) protecting group.

In some embodiments, the carboxylic acid protecting group ^P3 is selected from methyl, ethyl, tert-butyl, allyl (All), 2,4-dimethoxybenzyl (Dmb), 9-fluorenylmethyl (Fm), benzyl (Bn). In some embodiments, ^P3 is an allyl (All) protecting group.

In some embodiments, ^P4 is a trityl protecting group.

In some embodiments, the subunits of the polyamino acid synthon have a D-configuration, for example, the synthon is a D-enantiomer. In some embodiments, the polyamino acid synthon having a D-configuration subunit can be represented by the following formula:

In some embodiments, the subunits of the polyamino acid synthon have an L-configuration, for example, the synthon is an L-enantiomer. In some embodiments, the polyamino acid synthon having an L-configuration subunit can be represented by the following formula:

In some embodiments, the subunits of the polyamino acid synthon have both a D-configuration and an L-configuration.

In some embodiments, the amino acid side chains of the linear peptide have protecting groups. In some embodiments, the amino acid side chain protecting groups are selected from 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), xanthene (Xan), 2,6-dichlorobenzyl (2,6-Cl ₂ Bzl) and 2-bromobenzyl carbonate (2-BrZ). In some embodiments, the amino acid side chain protecting group is tert-butyl (tBu) or trityl (Trt).

In some embodiments, the amino acid side chains of the linear peptide having protecting groups on their side chains are Cys ₁ , Glu ₃ , Cys ₅ , Cys ₆ , Asn ₇ , Tyr ₁₁ and Cys ₁₃ of SEQ ID NO: 1.

In some embodiments, the plurality of amino acids and synthons are coupled to form the linear peptide of step (i) by a carbodiimide-mediated reaction or by a reaction mediated by a non-carbodiimide coupling agent, 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(dimethyl-amino)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).

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

In some embodiments, the carbodiimide-mediated reaction mixture further comprises an amino acid racemization inhibitor. In some embodiments, the racemization inhibitor is selected from 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.

In some embodiments, the solvent for carbodiimide-mediated reactions is, but is not limited to, N-methylpyrrolidone (NMP), dichloromethane (DCM), chloroform, or dimethylformamide (DMF). In some embodiments, the solvent for carbodiimide-mediated reactions is N-methylpyrrolidone (NMP).

In some embodiments, pyridine is used in carbodiimide-mediated reactions to prevent premature cleavage of the linear peptide from the solid support.

In some embodiments, at least one amino acid is coupled via a non-carbodiimide coupling agent. In some embodiments, the non-carbodiimide coupling agent is TBTU.

The linear peptide of step (i) may be referred to as a "linear 12-mer" in the present application, and may be represented by the following formula:

(SEQ ID NO: 2) wherein the cystathionine thioether side chain bridge -CH ₂ -CH ₂ -S-CH ₂ - is composed of In some embodiments, the side chains of one or more underlined amino acids are protected. In some embodiments, the side chains of all underlined amino acids are protected.

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

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

After cleavage of the linear peptide from the resin to generate a protected peptide, in step (iii) 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 some embodiments, the protected peptide obtained in step (ii) is used as is in step (iii).

In some embodiments, the amino acid coupled to the C-terminus of the protected peptide is cysteine. In some embodiments, cysteine is protected. In some embodiments, the amino acid coupled to the C-terminus is S-trityl-L-cysteinyl-O-tert-butyl-ester. In some embodiments, the protected peptide obtained in step (ii) can be represented by the following formula:

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

After coupling 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.

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

Once the protecting groups are removed, the unprotected amine is coupled to the unprotected carboxylic acid group in step (v) to form the cyclized peptide.

After forming the cyclized peptide in step (v), the cyclized peptide is fully deprotected in step (vi) to obtain a fully deprotected peptide. In some embodiments, the fully deprotecting step (vi) comprises adding a mixture comprising at least ammonium iodide ( _NH4I ) and anisole.

Once the cyclized peptide is fully deprotected, the peptide is folded in step (vii) to form one or more additional cross-links to obtain the synthetic peptide: 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).

In some embodiments, the cross-linked synthetic peptide formed in step (vii) contains covalent bonds between the following amino acid residues of the synthetic peptide: Cys ₁ and Cys ₆ , Cth ₂ and Cys ₁₀ , and Cys ₅ and Cys _13. In some embodiments, the covalent bonds between Cys ₁ and Cys ₆ and Cys ₅ and Cys ₁₃ are disulfide bonds. In some embodiments, the covalent bond between Cth ₂ and Cys ₁₀ is a thioether bond.

After folding of the fully deprotected peptide, the synthetic peptide of SEQ ID NO: 1 was purified.

In some embodiments, the method includes the 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. In the case where 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.

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 a dilution step with an organic solvent mixture. In some embodiments, the organic solvent mixture comprises at least one of acetonitrile or methyl tert-butyl ether (MTBE).

Also described herein are methods for preparing synthetic peptides of Formula I:

The method comprises

(i) coupling a C-terminal resin-bound Tyr-Gly peptide in which the Tyr amino acid residue is protected to a polyamino acid synthon of formula II:

in:

^P1 and ^P2 are different amine protecting groups;

^P3 is a carboxylic acid protecting group; and

^P4 is a thiol protecting group,

To form a resin-bound peptide of formula III:

(ii) removing the ^P2 protecting group of formula III to obtain a resin-bound peptide of formula IV having an unprotected amine group:

(iii) coupling P ² -alanine to the resin-bound peptide of Formula IV via the free amine group of Formula IV to form the resin-bound peptide of Formula V:

(iv) removing the ^P2 protecting group of formula V to obtain a free amine group, and then coupling the free amine group to a ^P2 -amino acid,

wherein the side chains of the amino acids may be protected;

(v) repeating step (iv) six more times to form a resin-bound peptide of formula VI:

wherein at least one of the amino acid side chains is 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 cysteine,

wherein cysteine comprises a carboxylic acid protecting group, and

The side chain of cysteine amino acid can be protected.

To obtain the protected peptide of formula VII

wherein ^P5 is a carboxylic acid protecting group different from ^P3 ;

(viii) removing the ^P2 protecting group and the ^P3 protecting group to obtain a free amine group and a free carboxylic acid group;

(ix) coupling the free amine group with the free carboxylic acid group to obtain the cyclized peptide of formula VIII:

(x) fully deprotecting the cyclized peptide to obtain a fully deprotected peptide; and

(xi) folding the fully deprotected peptide by forming two disulfide bonds to obtain the synthetic peptide of formula I.

As used herein, a P ² -amino acid is an amino acid in which the amine group is protected with an amine protecting group that is different from the amine protecting group of P ^1. For example, P ² -alanine would have the following structural formula:

In some embodiments, the method further comprises acetylation of the free amine group in Formula I to obtain a synthetic peptide of Formula IX:

In some embodiments, ^P1 is an acetyl group and steps (i)-(ix) are performed as described above. However, in step (x) the acetyl group represented by ^P1 is not removed during the complete deprotection step, and the step (x) folding step forms a compound represented by Formula IX.

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 groups are selected from 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), xanthene (Xan), 2,6-dichlorobenzyl (2,6-Cl ₂ Bzl) and 2-bromobenzyl carbonate (2-BrZ). In some embodiments, the amino acid side chain protecting group is tert-butyl (tBu) or trityl (Trt).

In some embodiments, P1 and ^P2 are each a protecting group selected from fluorenylmethoxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), carboxybenzyl (Cbz) and allyloxycarbonyl (Alloc). In some embodiments, ^P1 is a tert-butyloxycarbonyl (Boc) protecting group. In some embodiments ^{, P2} is a fluorenylmethoxycarbonyl (Fmoc) protecting group.

In some embodiments, ^P3 is a protecting group selected from methyl, ethyl, tert-butyl, allyl (All), trityl, 2,4-dimethoxybenzyl (Dmb), 9-fluorenylmethyl (Fm) and benzyl (Bn). In some embodiments, ^P3 is an allyl (All) protecting group.

In some embodiments, ^P4 is a protecting group selected from acetamidomethyl (Acm), tert-butyl (t-Bu), 3-nitro-2-pyridylthioyl (NPYS), 2-pyridyl-thioyl (Pyr) and trityl (Trt). In some embodiments, ^P4 is a trityl protecting group. In some embodiments, ^P4 is a tert-butyl protecting group.

Compound

Also disclosed herein is a compound or a pharmaceutically acceptable salt thereof, which is represented by the following structural formula:

in:

^P1 and ^P2 are hydrogen or an amine protecting group, provided that when ^P1 and ^P2 are both amine protecting groups they are not the same amine protecting group;

^P3 is hydrogen or a carboxylic acid protecting group; and

^P4 is hydrogen or a thiol protecting group.

In some embodiments, at least one of P ¹ , P ² , P ³ and/or P ⁴ is hydrogen. In some embodiments, each of P ¹ to P ⁴ is hydrogen.

In some embodiments, ^P1 to ^P4 are each a protecting group (eg, ^P1 and ^P2 are each an amine protecting group, ^P3 is a carboxylic acid protecting group, and ^P4 is a thiol protecting group).

In some embodiments, ^P1 and ^P2 are each an amine protecting group selected from acetyl, fluorenylmethoxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), carboxybenzyl (Cbz) and allyloxycarbonyl (Alloc). In some embodiments, ^P1 is acetyl. In some embodiments, ^P1 is a tert-butyloxycarbonyl (Boc) protecting group. In some embodiments, ^P2 is a Fmoc protecting group.

In some embodiments, ^P3 is a carboxylic acid protecting group selected from methyl, ethyl, tert-butyl, allyl (All), trityl, 2,4-dimethoxybenzyl (Dmb), 9-fluorenylmethyl (Fm) and benzyl (Bn). In some embodiments, ^P3 is an allyl (All) protecting group.

In some embodiments, ^P4 is a thiol protecting group selected from acetamidomethyl (Acm), tert-butyl (t-Bu), 3-nitro-2-pyridylthiosulphide (NPYS), 2-pyridyl-thiosulphide (Pyr) and trityl (Trt). In some embodiments, ^P4 is a trityl protecting group.

In some embodiments, the compound or a pharmaceutically acceptable salt thereof is a compound of Formula A:

In some embodiments, to obtain the compound of Formula A, three building blocks (parts A-C) are synthesized separately and combined (part D) to synthesize the compound of Formula A, as shown in the following scheme:

Part A:

Part B:

Part C:

Part D:

In some embodiments, to obtain the compound of formula A, the diunit compound (Alloc-HCys((Fmoc-Ala-OH)3-yl)-OAll) is first synthesized as shown in the following scheme:

Example

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

Reagents and solvents

Tables 1-3 list the starting materials, reagents and solvents used to make the claimed peptides, respectively.

Table 1: List of starting materials

Table 2: Reagent List

Table 3: List of solvents

Example 1

Method for producing the 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 pharmaceutically acceptable salt thereof, is to be used in the planned clinical study and is manufactured in accordance with Good Manufacturing Practice (GMP) regulations. All abbreviations are listed in Table 2 (reagent list) and Table 3 (solvent list), respectively.

The peptide of SEQ ID NO: 1, the N-terminally modified peptide of SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof, is manufactured according to the synthetic scheme described below. The synthetic route combines the stepwise synthesis of parts of the peptide chain according to the well-established principles of solid phase peptide chemistry. This is followed by the following sequential steps: incorporation of 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.

An exemplary method for making a peptide of SEQ ID NO: 1, an N-terminally modified peptide of SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof is presented in Figure 1. All starting materials constituting the primary sequence of the peptide 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.

After the assembly is completed, the folding of two disulfide bridges is induced between the designated cysteine residues. The thioether bond between the homocysteine (position 2) and cysteine (position 10) side chains is preformed in the tripeptide building block. Subsequent cyclization is induced by forming a lactam bond, as described below (step 5). After the final chromatographic purification, the drug substance is isolated by precipitation and drying.

Peptide synthesis

Step 1: Stepwise solid phase assembly

The primary amino acid sequence was assembled by an iterative process starting from 2-chlorotrityl resin preloaded with glycine residues (Gly ₁₂ for synthetic peptides). Nine consecutive cycles detailed in Tables 4-5 were performed as follows:

- The N-terminal Fmoc protecting group was removed from the previously introduced amino acid residue by treatment with a base (piperidine) in dimethylformamide (DMF), followed by extensive washing with DMF. This deprotection was not performed after the last incorporation step (glutamic acid). The deprotection reaction was monitored by HPLC.

-exist The next Fmoc-protected amino acids or building blocks were coupled in NMP under the action of DIC in the presence of DIPEA, with appropriate protection on their relevant side chains, except for the first reaction (coupling of tyrosine to glycine anchored on the resin) which was carried out in NMP under the action of TBTU in the presence of DIPEA. The coupling reactions were monitored by HPLC.

-Wash extensively with DMF.

Table 4. SPPS cycles and coupling conditions.

Table 5. Detailed description of the steps in Table 4.

Coupling

NMP is used as a solvent for dissolving amino acids, removing Fmoc-solutions, dissolving coupling agents, blocking solutions and pyridine-kick. Pyridine-kick is to add 3.6eq of pyridine to each coupling cycle to avoid partial cleavage of growing peptides from solid supports. Pyridine-kick is used for all coupling reactions, except for the case of coupling cysteine residues as discussed below. DMF is only used for the washing steps after coupling and removing Fmoc steps. Because NMP is used instead of DMF, the coupling time for standard amino acids needs to be extended from 90min to 180min, and the coupling time for tripeptide building blocks needs to be extended from 180min to 360min. Only 1.2 equivalents of tripeptides are used to replace 2.0 equivalents of standard coupling. The conditions of the standard method are shown in Table 4. SPPS of protected linear peptides as described in the scheme (Table 5) is carried out on a Tribute automatic synthesizer with a 0.75mmol scale using preloaded H-Gly-CT resin. Pyridine-promotion was used to minimize premature cleavage of the peptide from the resin due to acidic coupling conditions.

Analytical HPLC LC-MS analysis showed no evidence of high levels of missing sequences. The experiments showed that 1.2 equivalents of the tripeptide building block were suitable for complete coupling.

Racemization of cysteine (pyridine-promoted)

SPPS using DIC/Oxyma, which causes acidic coupling conditions, can lead to partial cleavage of the peptide from the resin. Therefore, pyridine-promotion was established. As indicated above, pyridine-promotion is the addition of a portion of pyridine to drive the reaction to completion. It is known that alkaline coupling of cysteine can cause racemization. Therefore, pyridine-promotion is not implemented during the coupling of cysteine residues because they are particularly sensitive to alkaline conditions (e.g., induced by pyridine) associated with the risk of racemization. Therefore, a test peptide using methionine instead of the L-tripeptide was synthesized once with pyridine-promotion of cysteine coupling and once without pyridine-promotion of cysteine coupling.

The use of pyridine-promotion for all couplings resulted in a D-cysteine content of 1.49% and for peptides synthesized without pyridine-promotion for cysteine couplings, a D-cysteine content of 1.35%. Furthermore, there was no effect on the resin yield of SPPS. Overall, the effect on the yield and quality of the product obtained was minimal. However, in order to reduce the risk of racemization, pyridine-promotion was not used for couplings of cysteine and tripeptides (chemically similar to protected cysteine).

Resin Loading

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

SPPS operation

The build was run at 7.5 mmol scale on preloaded H-Gly-2CT resin at 0.65 mmol/g. Each coupling step was monitored by Kaiser test. The results are given in Table 6. A mild test cut was performed and analyzed by UHPLC. A purity of 87.3% AN was achieved. The yield on resin was 98%.

A second SPPS batch was run automatically. The scale was also 7.5 mmol and the resin loading was 0.69 mmol/g. A purity of 89.4% AN in quantitative (≥100%) yield on the resin was achieved.

Table 6. Kaiser test results for SPPS runs.

Step 2: Cleavage of the resin-peptide bond

The peptide was cleaved from the resin under mild acidic treatment retaining the side chain protecting groups as well as the N-αBoc protecting group of the Cys ₁ residue. The completeness of this reaction was time specific. The reaction mixture was concentrated by evaporation and the solvent was exchanged to DMF.

To avoid the formation of sticky solids that adhere to glassware, direct coupling of the C-terminal cysteine was tested. This omits a separation step, which results in shorter cycle times. The direct coupling was tested with a gentle cleavage batch, where different anti-solvents have been tested to precipitate the peptide. The batch was evaporated to an oil, reconstituted with DCM and evaporated again to remove any residual anti-solvent. After reconstitution with DCM, the peptide was used as is for cysteine coupling. Table 7 shows the cleavage conditions.

Table 7. Conditions for mild resin cleavage.

Step 3: Incorporation of the C-terminal residue and in situ deprotection of the protecting group from Glu ₃

Activate the peptide solution from the cutting of step 2 by DIC/HOPO, and carry out in DMF in the presence of DIEA as alkali with the coupling of H-Cys (Trt) -OtBu.When the reaction monitored by HPLC is complete, cut the Fmoc protecting group of N-αGlu ₃ residues by directly adding piperidines in the reaction mixture.After the reaction monitored by HPLC is complete, separate the product of reaction by extraction, precipitation, filtration and drying.Table 8 shows the cysteine coupling conditions.

Table 8. Cysteine coupling conditions.

Step 4: Removal of protecting groups from Hcy ₂

The O-allyl ester protecting group of C-αHcy ₂ was then removed by dissolving it in DCM and cleaving the O-allyl ester with a palladium-containing catalyst (Pd(PPh ₃ ) ₄ ) in the presence of phenylsilane as a scavenger. The progress of the reaction was monitored by HPLC. The deallylation conditions were as follows:

1) Dissolve Pd(PPh ₃ ) ₄ (0.05 eq) in DCM

2) Add phenylsilane (2 eq) and 13-mer to Pd(PPh ₃ ) ₄ /DCM solution

3) Stir for 1 h and monitor the reaction progress by HPLC

4) When deallylation is complete, the solution is ready for the next step.

Step 5: Cyclization via Hcy ₂ →Glu ₃ coupling

After step 4 was completed, HOPO and DIC were added directly to the reaction mixture to induce cyclization via coupling between the carboxyl function of Hcy ₂ and the amine function of Glu _3. The reaction was monitored by HPLC. Table 9 shows the cyclization conditions.

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

Cyclization was also tested at 100 mg/mL. To achieve this, the coupling agent was added directly to the deallylation solution. Judging from the chromatogram, there was no visible peak showing oligomerization. Two cyclization concentrations (50 and 100 mg/mL) gave very similar results. The higher concentration did not bring any benefit in terms of purity.

Table 9. Cyclization conditions.

Step 6: Complete deprotection

Pd removal

Once the cyclization is complete, the reaction mixture is concentrated by evaporation under vacuum (to about 50% of the original volume) and treated by silicon-thiol (a scavenger specifically for the removal of Pd-based catalysts). The complexed palladium-scavenger is filtered off and the collected filtrate is further concentrated by evaporation. Table 10 shows the Pd removal conditions.

Table 10. Pd removal conditions.

The still protected cyclized 13-mer peptide was then fully deprotected by adding a mixture of DTT, water, thioanisole, TIS and NH ₄ I in TFA. After the reaction was complete, the crude deprotected peptide was recovered by precipitation in n-heptane and MBTE. The obtained solid was dried under vacuum. Table 11 shows the full deprotection conditions.

Table 11. Conditions for complete deprotection.

Step 7: Folding via disulfide bridges

The crude deprotected peptide from step 6 was taken up in ammonium bicarbonate and DMSO was added to induce folding by forming two disulfide bridges between the side chains of Cys ₁ and Cys ₆ and Cys ₅ and Cys ₁₃ , respectively. The folding reaction was monitored by HPLC. Table 12 shows the conditions used for the folding step.

Table 12. Peptide folding conditions

Step 8: Primary (first) purification

After step 7 is completed, the reaction mixture is acidified with TFA, diatomaceous earth is added as a filter aid and the resulting slurry is filtered. The clarified filtrate is directly loaded on a preparative HPLC column filled with a C18 (3) stationary phase. Purification is performed by gradient elution in 0.1% TFA in acetonitrile and 0.1% TFA in acetonitrile: water (5:95 v/v). Individual fractions are collected and selected based on analytical HPLC monitoring: fractions showing a purity of ≥90% (area %) are combined.

Step 9: N-terminal acetylation

Direct acetylation of the folded peptides in the folding solution was tested. After adding 15 eq of AcOSu, only very little conversion was detected. After adjusting the pH and the additional charge of AcOSu, still no substantial conversion was obtained. This was repeated with the second folding solution with the same results. Adding Ac ₂ O did not cause a stronger conversion.

To get an impression of the acetylation reaction of this compound, two experiments were run overnight using 15 eq AcOSu and 2 eq Ac ₂ O. Both reactions showed promising results, with the reaction with AcOSu not being complete after 15 h.

The reaction was repeated with increasing equivalents of _Ac2O (3eq and 4eq) and monitored after 18h. Higher amounts of acetylating agent appeared to drive the reaction to completion, and the shortened reaction time appeared to reduce the amount of side reactions, especially for the two largest late eluting impurities.

Therefore, the final method was limited to runs with 4.0 eq Ac ₂ O. Therefore, the acetylation step 9 was run from the pool collected from the primary (first) purification step 8, which was diluted in ammonium bicarbonate solution containing DMSO and then the N-αCys ₁ residue was acetylated by acetic anhydride. The reaction was monitored by HPLC. Table 13 shows the conditions for the acetylation step.

Table 13. N-terminal acetylation conditions

Step 10: Secondary purification

The acetylation reaction mixture was diluted 1/1 in ammonium acetate solution, the pH was adjusted to 7-8 with a 30% (v/v) aqueous solution of ammonium hydroxide, and final purification was performed by injection on a preparative HPLC column packed with a C18(3) stationary phase. Purification was performed by gradient elution in acetonitrile and 25 mM aqueous ammonium acetate:acetonitrile (95:5, v/v).

Individual fractions were collected and selected based on analytical HPLC monitoring: fractions showing a purity > 95% (area %) and no single impurity > 1.0% (area %) were combined.

In cases where the acetylation conditions were optimized to reduce the starting material to less than 0.1%, a second purification provided a slight polish of the material and facilitated the ion exchange from trifluoroacetate to acetate.

Step 11: Precipitation, Isolation by Filtration and Drying

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

The solid was wetted with 30% (v/v) aqueous acetonitrile followed by water and isolated by final drying under vacuum, which yielded the synthetic peptide of SEQ ID NO:1.

Table 14. Conditions for step 11.

Example 2

Preparation of Alloc-HCys(Fmoc-Ala-OH)-3-yl)-OAll

The following reaction scheme shows the preparation of the title compound:

Synthesis of Alloc-Hser-OAll

In a 5-liter three-necked flask, 500g of (S)-3-aminodihydrofuran-2(3H)-one hydrobromide was dissolved in 1L of water. A solution of 230g of NaOH in 1L of water was added. The reaction was monitored by TLC. After the reaction was complete, the pH value was adjusted to 7.5 by adding concentrated HCl. 230g of Na ₂ CO ₃ was added, and the mixture was cooled to 0° C. 331g of allyloxycarbonyl chloride (Alloc-Cl) was added dropwise while the temperature was maintained at 0-10° C. and the pH value was maintained at 7.5. The mixture was stirred overnight. The solvent was then removed, and 2500mL of DMF was then added. After the addition of 230g of NaHCO ₃ , 665g of allyl bromide was added. The mixture was stirred overnight, and 7000mL of water was then added to quench the reaction. The mixture was extracted with MTBE (5L*3), and the combined organic phase was washed with 800mL of NaHCO ₃ , 800mL of KHSO ₄ and 800mL of brine in sequence. After removing the solvent MTBE, the residue was purified by column chromatography (SiO ₂ ) to obtain 273.9g of an oily product. ¹ H NMR (400MHz, 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 calculated for C11H17NO5[M+H] ⁺ : 244.12; ESI-MS found: 244.08.

Synthesis of Alloc-Abu(4-Br)-OAll

In a 10L three-necked flask, 378g of Alloc-HSer-OAll and 216g of _Et3N were dissolved in 3400mL of DCM. Then 215g of methylsulfonyl chloride (MsCl) was added dropwise while the temperature was maintained at 0-10°C. After the reaction was complete, 3400mL of acetone was added and then 1356g of LiBr was added in batches. The reaction was maintained at 25-30°C overnight. 200g of LiBr and 520mL of acetone were added to drive the reaction to completion. The solvent was then removed and the residue was merged with another batch starting from 153g of Alloc-HSer-OAll. 572g of 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 calculated for C11H16BrNO4 [M+H] ⁺ : 306.03; ESI-MS found: 306.11.

Synthesis of (Fmoc-Cys-O(t-Bu)) ₂

Mix 210g of L-cysteine and 4kg of tert-butyl acetate in a 5L three-necked flask. Add 301.2g of HClO ₄ dropwise. Stir the mixture overnight. Add 2.3kg of K ₂ CO ₃ and 4.0kg of water. Stir the mixture overnight and then filter through 20g of diatomaceous earth. After separation, wash the organic layer with 1L of brine and then concentrate to provide 258.8g of oily residue. After dissolving the residue in 2400L of THF, add 430g of Fmoc-OSu. Then, add 126g of N-methylmorphine dropwise. Stir the reaction mixture for 2 hours. Remove the THF solvent and add 1500mL of DCM to dissolve the residue. The organic solution is washed with 10% citric acid solution, saturated NaHCO ₃ and brine in sequence. After removing the solvent, add 500mL of EtOAc. 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).

Synthesis of Fmoc-Cys-O(t-Bu)

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) ₃ P. After stirring the mixture for 2 hours, 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 organic matter was washed with 450 mL of 10% citric acid and 450 mL of brine in sequence. The organic solution was used directly in the next step.

Synthesis of Alloc-HCys((Fmoc-Ala-O(t-Bu))-3-yl)-OAll

In a 10L 4-necked flask, 1080 g of tetrabutylammonium bromide was dissolved in 3350 mL of saturated NaHCO _3. 500 mL of a solution of 257 g of Alloc-Abu(4-Br)-OAll in EtOAc and 2.8 L of the Fmoc-Cys-O(t-Bu) solution obtained in the previous step were added. The mixture was stirred overnight. After washing with 1 L of brine, the organic solution was concentrated. 418 g of an oily residue was obtained by column chromatography (SiO ₂ ). The yield was 80%. ¹ HNMR (400MHZ, chloroform-d) δ7.76 (d, J=7.5Hz, 2H), 7.61 (d, J=7.5Hz, 2H), 7.40 (t, J=7.4Hz, 2H), 7.31 (t, J=7.4Hz, 2H), 5.97-5.81 (m, 2H), 5.70 (d, J=7.8Hz, 1H), 5 .43(d, J＝8.3Hz, 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 calculated for C33H40N2O8S [M+Na] ⁺ : 647.24; ESI-MS found: 647.24.

Synthesis of Alloc-HCys((Fmoc-Ala-OH)-3-yl)-OAll

To a 5 L flask were added 308 g of Alloc-HCys((Fmoc-Ala-O(t-Bu)-3-yl)-OAll, 2430 mL of TFA and 53 g of i-Pr ₃ SiH. After the solution was stirred at 20-25° C. overnight, 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 solution was neutralized with 4 L of saturated NaHCO ₃ aqueous solution. After filtering the mixture, the solid was dissolved in 3 L of DCM. The organic solution was acidified and washed with 1 L of 20% citric acid solution. The resulting organic solution was washed with 900 mL of 10% citric acid solution and 500 mL of brine in sequence. 150 g of MgSO ₄ The organic layer was dried and the mixture was filtered. The solvent was removed and the residue was stirred until solidified to obtain 237 g of a solid product. The yield was 84.4%. ¹ H NMR (400MHz, chloroform-d) δ7.75 (d, J=7.5Hz, 2H), 7.60 (d, J=7.5Hz, 2H), 7.39 (t, J=7.5Hz, 2H), 7.30 (t, J=7.4Hz, 2H), 6.43 (s, 3H), 5.99-5.79 (m, 3H), 5.55 (d, J= 8.3Hz, 1H), 5.39-5.13 (m, 4H), 4.76-4.28 (m, 8H), 4.23 (dd, J=7.0Hz, 1H), 3.22-2.76 (m, 2H), 2.74-2.48 (m, 2H), 2.14 (s, 1H), 1.97 (s, 1H). is C29H32N2O8S[M+H] ⁺ Calculated ESI-MS: 569.20; Found ESI-MS: 569.12.

Example 3

Preparation of Fmoc-L-Cth[3-Boc-L-Cys(Trt),4-O-allyl]-OH building block

To obtain the Fmoc-L-Cth[3-Boc-L-Cys(Trt),4-Oallyl]-OH building block, three building blocks were synthesized individually (parts A-C) and combined (part D) to synthesize the title compound as shown in the following scheme:

Part A:

Part B:

Part C:

Part D:

Other Implementations

The present invention is not limited in scope by the specific embodiments described herein. In fact, various modifications of the present invention other than those described herein will become apparent to those skilled in the art from the foregoing description and drawings. Such modifications are intended to fall within the scope of the appended claims. It should also be understood that all values are approximate and are provided for description.

The disclosures of all patents, patent applications, publications, product specifications, and protocols cited throughout this application are incorporated herein by reference in their entirety for all purposes.

Claims (58)

1. A method of producing a synthetic peptide or a pharmaceutically acceptable salt thereof, the method comprising:

(i) Chemically synthesizing a linear peptide having its C-terminal end bound to a solid support using a plurality of amino acids and at least one polyamino acid synthon, the linear peptide having a protecting group in one or more amino acids and/or polyamino acid synthons;

wherein at least one amine group of the polyamino acid synthon has a protecting group different from 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, wherein the amino acid has an unprotected amine group, a protected carboxylic acid group, and optionally a 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 the unprotected amine and an unprotected carboxylic acid group to form a cyclized peptide;

(vi) Fully deprotecting the cyclized peptide to obtain a fully deprotected peptide;

(vii) Folding the fully 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;

Wherein the synthetic peptide comprises the amino acid sequence:

Cys₁ Cth₂ Glu₃ Leu₄ Cys₅ Cys₆ Asn₇ Val₈ Ala₉ Cys₁₀ Tyr₁₁ Gly₁₂ Cys₁₃(SEQ ID NO:1); And

Wherein the synthetic peptide contains covalent bonds between the following amino acid residues of the synthetic peptide:

a) Cys ₁ and Cys ₆,

B) Cth ₂ and Cys ₁₀, and

C) Cys ₅ and Cys ₁₃.

2. The method of claim 1, further comprising:

Precipitating the synthetic peptide from the solution.

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

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

5. The method of any one of claims 1-4, wherein the polyamino acid synthon is a compound represented by the formula:

Wherein:

p ¹ and P ² are different amine protecting groups;

P ³ is a carboxylic acid protecting group; and

P ⁴ is a thiol protecting group.

6. The method of any one of claims 1-5, wherein the protecting group is selected from fluorenylmethoxycarbonyl (Fmoc), t-butoxycarbonyl (Boc), carboxybenzyl (Cbz), trityl, methyl, ethyl, t-butyl, allyl, 2, 4-dimethoxybenzyl (Dmb), 9-fluorenylmethyl (Fm), benzyl (Bn), t-butyldimethylsilyl, allyloxycarbonyl (alloc), t-butoxycarbonyl, acetamidomethyl (Acm), 3-nitro-2-pyridylmethylene (NPYS), and 2-pyridylmethylene (Pyr).

7. The method of claim 5 or 6, wherein the P ¹ is a tert-butoxycarbonyl (Boc) protecting group and the P ² is a 9-fluorenylmethoxycarbonyl (Fmoc) protecting group.

8. The method of any one of claims 5-7, wherein the P ³ is an allyl protecting group.

9. The method of any one of claims 5-8, wherein the P ⁴ is a trityl protecting group.

10. The method of any one of claims 1-9, wherein a subunit of the polyamino acid synthon has a D-configuration.

11. The method of any one of claims 1-9, wherein a subunit of the polyamino acid synthon has an L-configuration.

12. The method of any one of claims 1-11, wherein the subunits of the polyamino acid synthon have both D-and L-configurations.

13. The method of any one of claims 1-12, wherein the protected carboxylic acid of step (iv) is derived from a protected carboxylic acid group of the polyamino synthon.

14. The method of any one of claims 1-12, wherein the unprotected carboxylic acid of step (v) is derived from an unprotected carboxylic acid group of the polyamino synthon.

15. The method of any one of claims 1-14, wherein the plurality of amino acids and synthons are coupled by a carbodiimide-mediated reaction or by a reaction mediated by a non-carbodiimide coupling agent that is: 1- [ bis (dimethylamino) methylene ] -1H-1,2, 3-triazolo [4,5-b ] pyridinium 3-oxide Hexafluorophosphate (HATU), (2- (1H-benzotriazol-1-yl) -1, 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-oxoethyleneamino-oxy) -dimethylamino-morpholinomethylene) ] methylamine (methanaminium) hexafluorophosphate (COMU), 1-cyano-2-ethoxy-2-oxoethyleneamino-oxy-tri-pyrrolidine-phosphonium hexafluorophosphate (PyOxim), benzotriazol-1-yloxy-tri-pyrrolidine-hexafluorophosphate (BOP), 3-oxo-7-pyrrolidino-hexafluoropropane (AOT) or (PYP-3-oxophosphine.

16. The method of any one of claims 1-15, wherein at least one amino acid and/or synthon from a plurality of peptides is coupled by a carbodiimide-mediated reaction to form the linear peptide of step (i).

17. The method of claim 15 or 16, wherein the carbodiimide is selected from the group consisting of Diisopropylcarbodiimide (DIC), dicyclohexylcarbodiimide (DCC), and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC).

18. The method of claim 17, wherein the carbodiimide is DIC.

19. The method of any one of claims 15-18, wherein the carbodiimide-mediated reaction further comprises an amino acid racemization inhibitor.

20. The method of claim 19, wherein 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.

21. The method of claim 20, wherein the racemization inhibitor is 2-cyano-2- (hydroxyimino) acetate.

22. The method of any one of claims 15-21, wherein the solvent for the carbodiimide-mediated reaction is N-methylpyrrolidone (NMP), dichloromethane (DCM), chloroform, or Dimethylformamide (DMF).

23. The method of any one of claims 15-22, wherein pyridine is used in the carbodiimide-mediated reaction to prevent premature cleavage of the linear peptide.

24. The method of any one of claims 1-15, wherein the at least one amino acid is coupled by a non-carbodiimide coupling agent.

25. The method of claim 24, wherein the non-carbodiimide coupling agent is TBTU.

26. The method of claim 11, wherein the protected peptide obtained in step (ii) is used as such in step (iii).

27. The method of any one of the preceding claims, wherein the linear peptide in step (ii) is cleaved from a solid support with a weak acid solution.

28. The method of claim 1, wherein the cyclized peptide of step (v) is formed in solution.

29. The process of any one of claims 1-28, wherein the complete deprotection step (vi) comprises adding a mixture comprising at least ammonium iodide (NH ₄ I) and anisole.

30. The method of claim 1, wherein the method comprises an optional step (viii) of modifying the N-terminus of the synthetic peptide with one or more chemical moieties.

31. The method of claim 30, wherein the modification is acetylation of the N-terminus of the synthetic peptide.

32. The method of claim 30 or 31, wherein the peptide of step (vii) is purified prior to modification step (viii).

33. The method of any one of claims 1-32, wherein the covalent bond between Cys ₁ and Cys ₆ and Cys ₅ and Cys ₁₃ is a disulfide bond.

34. The method of any one of claims 1-33, wherein the covalent bond between Cth ₂ and Cys ₁₀ is a thioether bond.

35. The method of claim 2, wherein the precipitation step comprises an acidification step followed by a dilution step with an organic solvent mixture.

36. The method of claim 35, wherein the organic solvent mixture comprises at least one of acetonitrile or methyl tert-butyl ether (MTBE).

37. A method of preparing a synthetic peptide of formula I:

It comprises the following steps:

(i) Coupling a Tyr-Gly peptide of a C-terminal binding resin in which the Tyr amino acid residue is protected to a polyamino acid synthon of formula II:

Wherein:

p ¹ and P ² are different amine protecting groups; p ³ is a carboxylic acid protecting group; and P ⁴ is a thiol protecting group to form a resin binding peptide of formula III:

(ii) Removing the P ² protecting group of formula III to obtain a resin-bound peptide of formula IV having an unprotected amine group:

(iii) Coupling P ² -alanine via a free amine group of formula IV to the resin binding peptide of formula IV to form a resin binding peptide of formula V:

(iv) Removing the P ² protecting group of formula V to obtain a free amine group, then coupling the free amine group to a P ² -amino acid,

Wherein the side chain of the amino acid is optionally protected;

(v) Repeating step (iv) five or more times to form a resin binding peptide of formula VI:

Wherein at least one amino acid side chain is 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,

Wherein the cysteine comprises a carboxylic acid protecting group, and

Wherein the side chain of the cysteine amino acid may be protected,

To obtain a protected peptide of formula VII

Wherein P ⁵ is a carboxylic acid protecting group different from P ³;

(viii) Removing the P ² protecting group and the P ³ protecting group to obtain a free amine group and a free carboxylic acid group;

(ix) Coupling the free amine groups and free carboxylic acid groups to obtain a cyclized peptide of formula VIII:

(x) Fully deprotecting the cyclized peptide to obtain a fully deprotected peptide; and

(Xi) Folding the fully deprotected peptide by formation of two disulfide bonds to obtain a synthetic peptide of formula I.

38. The method of claim 37, further comprising acetylating the free amine groups in formula I to obtain a synthetic peptide of formula IX:

39. The method of claim 37 or 38, wherein Glu, cys, cys, asn, gly and Cys residues of formula VII have side chain protecting groups.

40. The method of any one of claims 37-39, wherein each of P ¹ and P ² is a protecting group selected from fluorenylmethoxycarbonyl (Fmoc), t-butoxycarbonyl (Boc), carboxybenzyl (Cbz), and allyloxycarbonyl (Alloc).

41. The method of claim 40, wherein the P ¹ is a tert-butoxycarbonyl (Boc) protecting group.

42. The method of claim 40, wherein the P ² is fluorenylmethoxycarbonyl (Fmoc) protecting group.

43. The method of any one of claims 37-42, wherein the P ³ is a protecting group selected from the group consisting of methyl, ethyl, t-butyl, allyl, trityl, 2, 4-dimethoxybenzyl (Dmb), 9-fluorenylmethyl (Fm), and benzyl (Bn).

44. The method of claim 43, wherein said P ³ is an allyl protecting group.

45. The method of any one of claims 37-44, wherein the P ⁴ is a protecting group selected from acetamidomethyl (Acm), t-butyl (t-Bu), 3-nitro-2-pyridyloxythio (NPYS), 2-pyridyloxythio (Pys), and trityl (Trt).

46. The method of claim 45, wherein the P ⁴ is a trityl protecting group.

47. The method of claim 45, wherein said P ⁴ is a tert-butyl protecting group.

48. A compound or a pharmaceutically acceptable salt thereof, represented by the following structural formula:

Wherein:

P ¹ and P ² are each hydrogen or an amine protecting group, provided that when both 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; and

P ⁴ is hydrogen or a thiol protecting group.

49. The compound of claim 48, or a pharmaceutically acceptable salt thereof, wherein each of P ¹ and P ² is an amine protecting group selected from acetyl, fluorenylmethoxycarbonyl (Fmoc), t-butoxycarbonyl (Boc), carboxybenzyl (Cbz), and allyloxycarbonyl (Alloc).

50. The compound of claim 49, or a pharmaceutically acceptable salt thereof, wherein P ¹ is acetyl.

51. The compound of claim 49, or a pharmaceutically acceptable salt thereof, wherein the P ¹ is a t-butoxycarbonyl (Boc) protecting group.

52. The compound of claim 49, or a pharmaceutically acceptable salt thereof, wherein the P ² is an Fmoc protecting group.

53. The compound of any one of claims 48-52, or a pharmaceutically acceptable salt thereof, wherein the P ³ is a carboxylic acid protecting group selected from methyl, ethyl, t-butyl, allyl, trityl, 2, 4-dimethoxybenzyl (Dmb), 9-fluorenylmethyl (Fm), and benzyl (Bn).

54. The compound of claim 53, or a pharmaceutically acceptable salt thereof, wherein said P ³ is an allyl protecting group.

55. The compound of any one of claims 48-54, or a pharmaceutically acceptable salt thereof, wherein said P ⁴ is a thiol protecting group selected from acetamidomethyl (Acm), t-butyl (t-Bu), 3-nitro-2-pyridyloxythio (NPYS), 2-pyridin-yloxythio (Pyr), and trityl (Trt).

56. The compound of claim 55, or a pharmaceutically acceptable salt thereof, wherein the P ⁴ is a trityl protecting group.

57. The compound of claim 48, wherein at least one of P ¹、P²、P³ and/or P ⁴ is hydrogen.

58. The compound of claim 48, wherein each of said P ¹-P⁴ is hydrogen.