Process and intermediates for the preparation of

Abstract

The present invention provides novel intermediates and processes for the preparation of tilpotide or a pharmaceutically acceptable salt thereof.

Description

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 477,734, filed on December 29, 2022, the entire contents of which are incorporated herein by reference.

[0003] Sequence Listing

[0004] This application contains a sequence listing that has been submitted electronically in XML format, the entire contents of which are incorporated herein by reference. This XML copy was created on December 27, 2023, is named 02021-0166-00304_SL.XML, and is 78,828 bytes in size.

[0005] Public content

[0006] The present disclosure provides processes and intermediates for preparing a GIP / GLP1 dual agonist peptide (referred to herein as "tirzepatide") or a pharmaceutically acceptable salt thereof.

[0007] Diabetes is a chronic disease characterized by hyperglycemia due to defects in insulin secretion, insulin action, or both. In type 2 diabetes ("T2D"), the combined effects of impaired insulin secretion and insulin resistance are associated with elevated glucose levels. The GIP / GLP1 dual agonist tilportide ("TZP") is described and claimed in U.S. Patent No. 9,474,780. Tilportide can be used to treat T2D. The entire contents of U.S. Patent No. 9,474,780 are incorporated herein by reference.

[0008] The large-scale production of pharmaceutically acceptable tilpotide presents numerous technical challenges that may affect overall yield and purity. Process and intermediate development is needed to provide improved or alternative methods for producing tilpotide. Similarly, efficient and environmentally friendly "green" processes, including stable intermediates, are needed to produce tilpotide with fewer purification steps. Improved or alternative technologies are also needed to provide tilpotide production processes with minimal waste streams, thereby improving environmental and operator safety. Furthermore, there is a need to avoid the use of transition metals and / or harsh reaction conditions that are incompatible with peptide synthesis.

[0009] The present disclosure addresses these needs by providing novel intermediates and processes for preparing telpotide (SEQ ID NO: 1) or a pharmaceutically acceptable salt thereof. The disclosed process for preparing telpotide provides intermediates and process reactions that embody several technological advances, including a highly efficient, reduced-step route while maintaining high quality and purity. Furthermore, the novel process and intermediates reduce resource intensity and minimize waste streams.

[0010] The novel processes described herein provide various embodiments of intermediates for the production of telpotide. Summary of the Invention

[0011] The present disclosure describes methods for preparing tilpotide or a pharmaceutically acceptable salt thereof.

[0012] In certain embodiments, the present disclosure describes a method for preparing telpol peptide or a pharmaceutically acceptable salt thereof, the method comprising: (a) coupling a peptide of SEQ ID NO: 10 with SEQ ID NO: 11 to form a peptide of SEQ ID NO: 12; and (b) deprotecting the peptide of SEQ ID NO: 12 to obtain telpol peptide or a pharmaceutically acceptable salt thereof. In certain embodiments, the peptide of SEQ ID NO: 12 obtained in step (a) is isolated prior to the deprotection step (b). In certain embodiments, the peptide of SEQ ID NO: 12 obtained in step (a) is washed prior to the deprotection step (b). In certain embodiments, the peptide of SEQ ID NO: 12 obtained in step (a) is washed after the deprotection step (b).

[0013] In certain embodiments, the present disclosure describes a method for preparing telpotide or a pharmaceutically acceptable salt thereof, the method comprising: (a) coupling a peptide of SEQ ID NO: 13 with SEQ ID NO: 14 to form a peptide of SEQ ID NO: 12; and (b) deprotecting the peptide of SEQ ID NO: 12 to obtain telpotide or a pharmaceutically acceptable salt thereof. In certain embodiments, the peptide of SEQ ID NO: 12 obtained in step (a) is isolated prior to step (b). In certain embodiments, the peptide of SEQ ID NO: 12 obtained in step (a) is washed prior to the deprotection step (b). In certain embodiments, the peptide of SEQ ID NO: 12 obtained in step (a) is washed after the deprotection step (b).

[0014] In certain embodiments, the present disclosure describes a method for preparing telpol peptide or a pharmaceutically acceptable salt thereof, the method comprising: (a) coupling a peptide of SEQ ID NO: 15 with SEQ ID NO: 16 to form a peptide of SEQ ID NO: 12; and (b) deprotecting the peptide of SEQ ID NO: 12 to obtain telpol peptide or a pharmaceutically acceptable salt thereof. In certain embodiments, the peptide of SEQ ID NO: 12 obtained in step (a) is isolated prior to step (b). In certain embodiments, the peptide of SEQ ID NO: 12 obtained in step (a) is washed prior to deprotection step (b). In certain embodiments, the peptide of SEQ ID NO: 12 obtained in step (a) is washed after deprotection step (b).

[0015] In certain embodiments, the present disclosure describes a compound of SEQ ID NO: 10 or a pharmaceutically acceptable salt thereof. In certain embodiments, the present disclosure describes a compound of SEQ ID NO: 10 or a pharmaceutically acceptable salt thereof, wherein the compound comprises one or more protecting groups. In certain embodiments, the one or more protecting groups are selected from Fmoc, Boc, tert-butyl and trityl. In certain embodiments, the compound does not comprise one or more protecting groups of SEQ ID NO: 10 (i.e., one or more protecting groups are removed from SEQ ID NO: 10). In certain embodiments, the compound does not comprise any protecting group in SEQ ID NO: 10 (i.e., all protecting groups are removed from SEQ ID NO: 10).

[0016] In certain embodiments, the present disclosure describes a compound of SEQ ID NO: 13, or a pharmaceutically acceptable salt thereof. In certain embodiments, the present disclosure describes a compound of SEQ ID NO: 13, or a pharmaceutically acceptable salt thereof, wherein the compound comprises one or more protecting groups. In certain embodiments, the one or more protecting groups are selected from Fmoc, Boc, tert-butyl, and trityl.

[0017] In certain embodiments, the present disclosure describes a compound of SEQ ID NO: 14, or a pharmaceutically acceptable salt thereof. In certain embodiments, the present disclosure describes a compound of SEQ ID NO: 14, or a pharmaceutically acceptable salt thereof, wherein the compound comprises one or more protecting groups. In certain embodiments, the one or more protecting groups are selected from Fmoc, Boc, tert-butyl, and trityl.

[0018] In certain embodiments, the present disclosure describes SEQ ID NO:15 compound or its pharmaceutically acceptable salt.In certain embodiments, the present disclosure describes SEQ ID NO:15 compound or its pharmaceutically acceptable salt, wherein the compound comprises one or more protecting groups.In certain embodiments, the one or more protecting groups are selected from Fmoc, Boc, tert-butyl and trityl.In certain embodiments, the compound does not comprise one or more protecting groups in SEQ ID NO:15 (i.e., from SEQ ID NO:15 remove one or more protecting groups).In certain embodiments, the compound does not comprise any protecting group in SEQ ID NO:15 (i.e., from SEQ ID NO:15 remove all protecting groups).

[0019] In certain embodiments, the present disclosure describes SEQ ID NO:16 compound or its pharmaceutically acceptable salt.In certain embodiments, the present disclosure describes SEQ ID NO:16 compound or its pharmaceutically acceptable salt, wherein the compound comprises one or more protecting groups.In certain embodiments, the one or more protecting groups are selected from Fmoc, Boc, tert-butyl and trityl.In certain embodiments, the compound does not comprise SEQ ID NO:16 one or more protecting groups (i.e., from SEQ ID NO:16 remove one or more protecting groups).In certain embodiments, the compound does not comprise SEQ ID NO:16 any protecting group (i.e., from SEQ ID NO:16 remove all protecting groups).

[0020] In certain embodiments, the present disclosure describes methods of preparing compounds of formula (I):

[0021]

[0022] wherein PG is a protecting group, the method comprising:

[0023] (a) contacting the compound of formula (Ia) with diisopropylethylamine

[0024]

[0025] (b) contacting the product of step (a) with 2-(5-norbornene-2,3-dicarboximido)-1,1,3,3-tetramethyluronium tetrafluoroborate in DMF; and

[0026] (c) adding the compound of formula (Ib) to the mixture of (b),

[0027]

[0028] In certain embodiments, the protecting group is selected from Boc and Fmoc. In certain embodiments, the protecting group is Fmoc. In certain embodiments, the method is carried out using continuous flow. In certain embodiments, the method is carried out in a flow reactor.

[0029] In certain embodiments, the present disclosure describes a method comprising filtering tilportide or a pharmaceutically acceptable salt thereof under one or more of the following conditions: (i) a temperature of 10 to 34° C., optionally about 20° C.; (ii) a turbulent flow / crossflow velocity of 1.94 to 2.46 m / s, optionally about 2.2 m / s; (iii) a laminar flow / shear rate of 2.1×10 3 to 2.7×10 3 1 / s, optionally about 2.4×10 3 1 / s; (iv) a primary product concentration of 32.8 to 47.2 mg / mL, optionally about 40 mg / mL; and (v) a secondary product concentration of 37 to 53 mg / mL, optionally about 45 mg / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1A 、 Figure 1B and Figure 1C Representative XRPD patterns of Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) pentamer Form A, Form C, and Form D are shown, respectively.

[0032] Figure 2A 、 Figure 2B 、 Figure 2C and Figure 2D Viscosity measurement results from gelation studies of peptide fragments of the present disclosure are shown. Figure 2A Viscosity measurement results from gelation studies related to process solvents are shown. Figure 2B Viscosity measurements from gelation studies are shown in relation to peptide concentration and gelation time. Figure 2C and Figure 2D Viscosity measurements from gelation studies are shown in relation to peptide concentration and processing shear rate. Detailed Description of the Invention

[0034] The present invention provides a method for preparing tilportide or a pharmaceutically acceptable salt thereof. In some embodiments, the method comprises step (a): coupling the peptide of SEQ ID NO: 10 with the peptide of SEQ ID NO: 11 to form the peptide of SEQ ID NO: 12.

[0035] The present invention also provides a method for preparing tilportide or a pharmaceutically acceptable salt thereof, comprising the step (a) coupling the peptide of SEQ ID NO: 13 with the peptide of SEQ ID NO: 14 to form the peptide of SEQ ID NO: 12.

[0036] The present invention also provides a method for preparing tilportide or a pharmaceutically acceptable salt thereof, comprising the step (a) coupling the peptide of SEQ ID NO: 15 with the peptide of SEQ ID NO: 16 to form the peptide of SEQ ID NO: 12.

[0037] In some embodiments, the method further comprises the step (b) deprotecting the peptide of SEQ ID NO: 12 to obtain telpotide or a pharmaceutically acceptable salt thereof.

[0038] In some embodiments, the method comprises isolating the coupled product of step (a) before performing the deprotection step (b). In some embodiments, the method comprises washing the coupled product of step (a) before performing the deprotection step (b). In some embodiments, the method comprises washing the coupled product of step (a) after performing the deprotection step (b).

[0039] Another aspect of the present disclosure provides a compound of SEQ ID NO: 10 or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of SEQ ID NO: 10 comprises one or more protecting groups.

[0040] Another aspect of the present disclosure provides a compound of SEQ ID NO: 13 or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of SEQ ID NO: 13 comprises one or more protecting groups.

[0041] Another aspect of the present disclosure provides a compound of SEQ ID NO: 14 or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of SEQ ID NO: 14 comprises one or more protecting groups.

[0042] Another aspect of the present disclosure provides a compound of SEQ ID NO: 15 or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of SEQ ID NO: 15 comprises one or more protecting groups.

[0043] Another aspect of the present disclosure provides a compound of SEQ ID NO: 16 or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of SEQ ID NO: 16 comprises one or more protecting groups.

[0044] In some embodiments, the one or more protecting groups are selected from Fmoc, Boc, tert-butyl, and trityl, or a combination thereof. In some embodiments, the one or more protecting groups are Fmoc. In some embodiments, the one or more protecting groups are Boc. In some embodiments, the one or more protecting groups are tert-butyl. In some embodiments, the one or more protecting groups are trityl. In some embodiments, the compound is protected with Fmoc, Boc, tert-butyl, and trityl. In some embodiments, the compound is protected with an Fmoc group. In some embodiments, the compound is protected with an Fmoc and Boc groups. In some embodiments, the compound is protected with Fmoc and tert-butyl. In some embodiments, the compound is protected with Fmoc and trityl. In some embodiments, the compound is protected with a Boc group. In some embodiments, the compound is protected with Boc and tert-butyl. In some embodiments, the compound is protected with Boc and trityl. In some embodiments, the compound is protected with tert-butyl. In some embodiments, the compound is protected with tert-butyl and trityl. In some embodiments, the compound is protected with trityl. In some embodiments, the compound is protected with Fmoc, Boc, and tert-butyl. In some embodiments, the compound is protected with Fmoc, Boc, and trityl. In some embodiments, the compound is protected with Boc, tert-butyl, and trityl. In some embodiments, the compound is protected with Fmoc, tert-butyl, and trityl. In some embodiments, the compound is protected with Fmoc, tert-butyl, and trityl.

[0045] In some embodiments, one or more protecting groups are removed in step (b). In some embodiments, all protecting groups are removed in step (b).

[0046] In another aspect of the present disclosure, a method for preparing telpotide side chain + lysine of formula (I) is provided:

[0047]

[0048] Wherein PG is a protecting group.

[0049] In some embodiments, the compound of formula (I) comprises two protecting groups. In some embodiments, the compound of formula (I) comprises one protecting group. In some embodiments, the compound of formula (I) comprises no protecting groups. In some embodiments, the compound of formula (I) is used to prepare telpotide. In some embodiments, the compound of formula (I) is used to prepare telpotide by a linear SPPS method.

[0050] In some embodiments, the method of preparing a compound of formula (I) comprises step (a): contacting a compound of formula (Ia) with diisopropylethylamine:

[0051]

[0052] Wherein PG is a protecting group.

[0053] In some embodiments, the method for preparing the compound of formula (I) further comprises step (b): contacting the product of step (a) with 2-(5-norbornene-2,3-dicarboximido)-1,1,3,3-tetramethyluronium tetrafluoroborate in DMF. In some embodiments, the method comprises step (c): adding a protected lysine of formula (Ib) to the mixture of step (b):

[0054]

[0055] In some embodiments, the protecting group of the compound of formula (I) is selected from Boc and Fmoc. In some embodiments, the protecting group of the compound of formula (I) is Boc. In some embodiments, the protecting group of the compound of formula (I) is Fmoc.

[0056] In some embodiments, the method of preparing a compound of formula (I) is performed using continuous flow. In some embodiments, the method of preparing a compound of formula (I) is performed in a flow reactor.

[0057] As used herein, the following abbreviations have the meanings set forth herein: “AEEA” means 17-amino-10-oxo-3,6,12,15-tetraoxa-9-azaheptadecanoic acid; “API” means active pharmaceutical ingredient; “CTC” means chlorotrityl; “DIC” means diisopropylcarbodiimide; “DCC” means dicyclohexylcarbodiimide; “DCM” means dichloromethane; “DCU” means dicyclohexylurea; “DIEA” means N,N-diisopropylethylamine; “DMF” means dimethylformamide; “DTT” means dithiothreitol; “Fmoc” means fluorenylmethoxycarbonyl chloride; “HATU” means (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate; “HFIP” means “IPA” denotes hexafluoroisopropanol; “IPA” denotes isopropanol; “L-GSH” denotes L-glutathione reducing solution; “LPPS” denotes liquid-phase peptide synthesis; “MTBE” denotes methyl tert-butyl ether; “OXYMA” denotes ethyl cyanohydroxyiminoacetate; “Pip” denotes piperidine; “SPPS” denotes solid-phase peptide synthesis; “TFA” denotes trifluoroacetic acid; “TFET” denotes 2,2,2-trifluoroethanethiol; “TIPS” denotes triisopropylsilane; “TCEP” denotes tris(2-carboxyethyl)phosphine; “TMSA” denotes trimethylsilylacetamide; “TNTU” denotes 2-(5-norbornene-2,3-dicarboximido)-1,1,3,3-tetramethyluronium tetrafluoroborate; “TZP” denotes tilportide; and “UPLC” denotes ultra-performance liquid chromatography.

[0058] As shown herein, the single-letter amino acid abbreviations are shown in bold, while atoms are shown in non-bold to distinguish them from the single-letter amino acid abbreviations. As used herein, when an amino acid abbreviation is preceded by a number, the number refers to the corresponding amino acid position in the final telportipeptide product. These numbers are provided for convenience, and the presence or absence of these numbers in the sequence does not affect the amino acid sequence or the peptide shown in the sequence.

[0059] As used herein, the term "protected" means that a protecting group is attached to a designated position. Those skilled in the art will recognize that various protecting groups are well known and that alternative protecting groups may be suitable for a particular process.

[0060] As used herein, the term "protecting group" or "amino acid protecting group" refers to a group that protects the acid portion or amine portion of an amino acid or a reactive portion on an amino acid side chain. "Acid moieties" include, for example, carboxylic acid groups (-COOH). "Amine moieties" include, for example, primary amine groups (-NH2), secondary amine groups (-NH-), amide groups (-C(O)-NH2), and guanidine groups ([-NHC(NH2)-NH2] +The acid or amine moiety can be part of a terminal amino acid in a peptide or polypeptide, or can be part of a side chain of a non-terminal amino acid in a peptide or polypeptide. Other reactive moieties on amino acid side chains include, for example, hydroxyl (-OH) and thiol (-SH) groups.

[0061] The protecting group may be a removable group known in the art, which serves to: (i) protect a reactive group (such as an amine or carboxylic acid group) from undesirable reactions during synthesis, for example, to block or protect the functionality of the reactive group when performing reactions involving other functional sites of the compound; and (ii) selectively deprotect in a multi-protected structure without affecting other protecting groups. Suitable protecting groups and methods for introducing and removing such protecting groups include methods known in the art, such as TW Green and PGM Wuts, Greene's Protective Groups in Organic Synthesis, John Wiley and Sons, 2007 and Isidro-Llobet et al., Amino Acid-Protecting Groups, Chem. Rev, 2009, 109(6), 2455-2504, the entire contents of which are incorporated herein.

[0062] Suitable protecting groups for aspartic acid (Asp) include, but are not limited to, tert-butyl (t-Bu), 3-methyl-3-pentyl (mpe), allyl, and 4-{N-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl]amino}benzyl (DMAB). In some embodiments, the protecting group for aspartic acid (Asp) is t-Bu or mpe.

[0063] Suitable protecting groups for serine (Ser), threonine (Thr) or tyrosine (Tyr) include, but are not limited to, t-Bu and triphenylmethyl (trityl or trt). In some embodiments, the protecting group for serine (Ser), threonine (Thr) or tyrosine (Tyr) is t-Bu or TRT.

[0064] Suitable protecting groups for glutamic acid (Glu) include, but are not limited to, t-Bu, trt, allyl, and DMAB. In some embodiments, the protecting group for glutamic acid (Glu) is t-Bu or trt.

[0065] Suitable protecting groups for glutamine (Gln) include, but are not limited to, trt, 4-methoxytrityl (4-methyltrityl, or MTT), acetamidomethyl (ACM), and trimethoxybenzyl (TMOB). In some embodiments, the protecting group for glutamine (Gln) is TRT.

[0066] Suitable protecting groups for lysine (Lys) include, but are not limited to, tert-butoxycarbonyl (Boc), allyloxycarbonyl (Alloc), 4-phenylacetyloxybenzyloxycarbonyl (PhAc), MTT, 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (ivDde), and 2-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde). In some embodiments, the protecting group for lysine (Lys) is Boc, MTT, or Alloc.

[0067] Suitable protecting groups for tryptophan (Trp) include, but are not limited to, Boc and formyl. In some embodiments, the protecting group for tryptophan (Trp) is Boc.

[0068] Suitable protecting groups for histidine (His) include, but are not limited to, Boc, trt, and 2,4-dinitrophenyl (dnp). In some embodiments, the protecting group for histidine (His) is Boc, trt, or dnp.

[0069] Exemplary acid protecting groups include esters such as substituted and unsubstituted C1-C8 lower alkyl (e.g., methyl, ethyl, tert-butyl), methoxymethyl, methylthiomethyl, 2,2,2-trichloroethyl, tetrahydropyranyl, substituted and unsubstituted phenylalkyl (e.g., benzyl) and substituted derivatives thereof (e.g., alkoxybenzyl, nitrobenzyl), cinnamyl, dialkylaminoalkyl (e.g., dimethylaminoethyl), trimethylsilyl, substituted and unsubstituted amides and hydrazides (e.g., amides and hydrazides of N,N-dimethylamine), 7-nitroindole, hydrazine, N-phenylhydrazine, acyloxyalkyl (e.g., pivaloyloxymethyl, propionyloxymethyl), aroyloxyalkyl (e.g., benzoyloxyethyl), alkoxycarbonylalkyl (e.g., methoxycarbonylmethyl), cyclohexyloxycarbonylmethyl, alkoxycarbonyloxy alkyl)-2-oxo-1,3-dioxol-4-yl)alkyl (e.g., (5-tert-butyl-2-oxo-1,3-dioxol-4-yl)methyl) and (5-phenyl-2-oxo-1,3-dioxol-4-yl)alkyl (e.g., (5-phenyl-2-oxo-1,3-dioxol-4-yl)methyl).

[0070] Exemplary amine and / or amide protecting groups include, but are not limited to, acyl (e.g., formyl, acetyl, chloroacetyl, trichloroacetyl, o-nitrophenylacetyl, o-nitrophenoxyacetyl, trifluoroacetyl, acetoacetyl, 4-chlorobutyryl, isobutyryl, o-nitrocinnamoyl, picolinyl, acylisothiocyanate, aminocaproyl, benzoyl), acyloxy (e.g., methoxycarbonyl, 9-fluorenylmethoxycarbonyl, 2,2,2-trifluoroethoxycarbonyl, 2-trimethylsilylethoxy-carbonyl, vinyloxycarbonyl, allyloxycarbonyl, tert-butoxycarbonyl (Boc), 1,1-dimethyl-propynyloxycarbonyl, benzyloxycarbonyl (Cbz), p-nitrobenzyloxycarbonyl, 2,4-dichlorobenzyloxycarbonyl), 9-xanthenyl, and trityl. Other exemplary amide protecting groups include, but are not limited to, o-nitrocinnamoyl, picolinoyl, aminocaproyl, benzoyl, acyloxy (e.g., methoxy-carbonyl, 9-fluorenylmethoxycarbonyl, 2,2,2-trifluoroethoxycarbonyl, 2-trimethylsilylethoxy-carbonyl, vinyloxycarbonyl, allyloxycarbonyl, tert-butoxycarbonyl (Boc), 1,1-dimethyl-propynyloxycarbonyl, benzyloxycarbonyl (Cbz), p-nitrobenzyloxycarbonyl, and 2,4-dichloro-benzyloxycarbonyl). Exemplary indole protecting groups include, but are not limited to, formyl (For) and tert-butoxycarbonyl (Boc). Exemplary imidazole protecting groups include, but are not limited to, tosyl (Tos), benzyloxymethyl (Bom), trityl (Trt), and tert-butoxycarbonyl (Boc). Exemplary guanidine protecting groups include, but are not limited to, 2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl (Pbf) and tert-butoxycarbonyl (Boc).

[0071] Exemplary hydroxy protecting groups include, but are not limited to, unsubstituted or substituted alkyl (e.g., tert-butyl, allyl, benzyl, methoxymethyl, tetrahydropyranyl, o-nitrobenzyl), silyl (e.g., tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), acyl (e.g., acetyl, benzoyl, pivaloyl). Exemplary thiol protecting groups include, but are not limited to, p-methylbenzyl (Meb), acetamidomethyl (Acm), and trityl (Trt).

[0072] Those skilled in the art will appreciate that a variety of resins are available for constructing the peptides described herein. For example, Sieber and Rink amide resins are well known to those skilled in the art for use in preparing the peptides described herein; however, other resins may also be selected for use in preparing the peptides described herein. Resins such as, but not limited to, 2-CTC and related resins may be used to prepare the target peptides, followed by a C-terminal amidation step.

[0073] The solid phase peptide synthesis (SPPS) construction described herein uses standard fluorenylmethoxycarbonyl chloride (Fmoc) peptide chemistry techniques and sequential couplings are performed using an automated peptide synthesizer. In some embodiments, the resin is swollen with DMF and then deprotected with 20% piperidine (Pip) / DMF (3×30 min). In some embodiments, subsequent Fmoc deprotection is treated with 20% Pip / DMF for 3×30 min, or 4×30 min for more difficult couplings. In some embodiments, after deprotection, the resin is washed with 10 volumes of DMF for 5×2 min. In some embodiments, amino acid preactivation is performed using a solution of diisopropylcarbodiimide (DIC) / ethyl cyanohydroxyiminoacetate (OXYMA) in DMF at room temperature for 30 min. In some embodiments, coupling of the activated amino acid to the resin-bound peptide occurs at a designated time for each individual amino acid. In some embodiments, each coupling is followed by a solvent wash with 10 volumes of DMF for 5×2 min. In some embodiments, to isolate the final product, the resin-bound product is washed with 10 volumes of DCM for 5 × 2 min to remove DMF. In some embodiments, the resin is washed with 10 volumes of IPA for 2 × 2 min to remove DCM, and then washed with 10 volumes of methyl tert-butyl ether (MTBE), and the product is then dried at 40 ° C under vacuum. In some embodiments, the resin-bound product is refrigerated (-20 ° C). In some embodiments, for analysis, the peptide is cleaved from the resin with an acidic mixture (mixture) consisting of trifluoroacetic acid (TFA) / H2O / TIPS (triisopropylsilane) / DTT (dithiothreitol) in the following ratios: (0.93v / 0.04v / 0.03v / 0.03w). In some embodiments, the resin is swollen with DCM (4-5 mL, 3 × 30 min) and drained. In some embodiments, the cleavage mixture (4-5 mL) is added to the pre-swollen resin, and the suspension is stirred at room temperature for 2 hours. In some embodiments, the solution is filtered, and the resin is then washed with a small amount of DCM and combined with the cleavage solution. In some embodiments, the resulting solution is poured into 7-10 times the volume of cold (0°C) methyl tert-butyl ether (MTBE). In some embodiments, the suspension is aged at 0°C for 30 min, the resulting precipitate is then centrifuged, and the clear solution is decanted. In some embodiments, the residue is suspended in an equal volume of MTBE, and the resulting suspension is then centrifuged again and decanted. In some embodiments, after decanting, the clear MTBE solution of the precipitated peptide is dried overnight at 40°C in a vacuum.

[0074] As described herein, native chemical ligation is a method for preparing full-length peptides containing cysteine or alanine in their sequences. This method utilizes the chemoselective reaction of two unprotected peptide fragments to produce a transient thioester-linked intermediate. This thioester-linked intermediate rearranges to form a full-length ligated product with a native peptide bond at the ligation site. Those skilled in the art will appreciate that native chemical ligation techniques can be used to chemically synthesize full-length peptides containing cysteine or alanine.

[0075] Listed implementation plans

[0076] Embodiment 1. A method for preparing tilpotide or a pharmaceutically acceptable salt thereof, the method comprising: (a) coupling a peptide of SEQ ID NO: 10 with SEQ ID NO: 11 to form a peptide of SEQ ID NO: 12; and (b) deprotecting the peptide of SEQ ID NO: 12 to obtain tilpotide or a pharmaceutically acceptable salt thereof.

[0077] Embodiment 2. The method of embodiment 1, wherein the peptide of SEQ ID NO: 12 from step (a) is isolated and then subjected to a deprotection step (b).

[0078] Embodiment 3. The method of embodiment 1 or 2, wherein the peptide of SEQ ID NO: 12 from step (a) is washed prior to the deprotection step (b).

[0079] Embodiment 4. The method of embodiment 1 or 2, wherein after the deprotection step (b), the peptide of SEQ ID NO: 12 from step (a) is washed.

[0080] Embodiment 5. A method for preparing tilpotide or a pharmaceutically acceptable salt thereof, the method comprising: (a) coupling a peptide of SEQ ID NO: 13 with SEQ ID NO: 14 to form a peptide of SEQ ID NO: 12; and (b) deprotecting the peptide of SEQ ID NO: 12 to obtain tilpotide or a pharmaceutically acceptable salt thereof.

[0081] Embodiment 6. The method of embodiment 5, wherein the peptide of SEQ ID NO: 12 from step (a) is isolated prior to step (b).

[0082] Embodiment 7. The method of embodiment 5 or 6, wherein the peptide of SEQ ID NO: 12 from step (a) is washed prior to the deprotection step (b).

[0083] Embodiment 8. The method of embodiment 5 or 6, wherein after the deprotection step (b), the peptide of SEQ ID NO: 12 from step (a) is washed.

[0084] Embodiment 9. A method for preparing tilpotide or a pharmaceutically acceptable salt thereof, the method comprising: (a) coupling a peptide of SEQ ID NO: 15 with SEQ ID NO: 16 to form a peptide of SEQ ID NO: 12; and (b) deprotecting the peptide of SEQ ID NO: 12 to obtain tilpotide or a pharmaceutically acceptable salt thereof.

[0085] Embodiment 10. The method of embodiment 9, wherein the peptide of SEQ ID NO: 12 from step (a) is isolated prior to step (b).

[0086] Embodiment 11. The method of embodiment 9 or 10, wherein the peptide of SEQ ID NO: 12 from step (a) is washed prior to the deprotection step (b).

[0087] Embodiment 12. The method of embodiment 9 or 10, wherein after the deprotection step (b), the peptide of SEQ ID NO: 12 from step (a) is washed.

[0088] Embodiment 13. The compound of SEQ ID NO: 10 or a pharmaceutically acceptable salt thereof, wherein the compound comprises one or more protecting groups.

[0089] Embodiment 14. The compound of Embodiment 13, wherein the one or more protecting groups are selected from Fmoc, Boc, tert-butyl, and trityl.

[0090] Embodiment 15. The compound of Embodiment 13 or 14, wherein the compound does not comprise one or more protecting groups of SEQ ID NO: 10 (ie, one or more protecting groups are removed from SEQ ID NO: 10).

[0091] Embodiment 16. The compound of embodiment 13 or 14, wherein the compound does not comprise any protecting groups of SEQ ID NO: 10 (ie, all protecting groups are removed from SEQ ID NO: 10).

[0092] Embodiment 17. The compound of SEQ ID NO: 13 or a pharmaceutically acceptable salt thereof, wherein the compound comprises one or more protecting groups.

[0093] Embodiment 18. The compound of Embodiment 17, wherein the one or more protecting groups are selected from Fmoc, Boc, tert-butyl and trityl groups.

[0094] Embodiment 19. The compound of SEQ ID NO: 14 or a pharmaceutically acceptable salt thereof, wherein the compound comprises one or more protecting groups.

[0095] Embodiment 20. The compound of Embodiment 19, wherein the one or more protecting groups are selected from Fmoc, Boc, tert-butyl and trityl groups.

[0096] Embodiment 21. The compound of SEQ ID NO: 15, or a pharmaceutically acceptable salt thereof, wherein the compound comprises one or more protecting groups.

[0097] Embodiment 22. The compound of Embodiment 21, wherein the one or more protecting groups are selected from Fmoc, Boc, tert-butyl and trityl groups.

[0098] Embodiment 23. The compound of Embodiment 21 or 22, wherein the compound does not comprise one or more protecting groups of SEQ ID NO: 15 (ie, one or more protecting groups are removed from SEQ ID NO: 15).

[0099] Embodiment 24. The compound of embodiment 21 or 22, wherein the compound does not comprise any protecting groups of SEQ ID NO: 15 (ie, all protecting groups are removed from SEQ ID NO: 15).

[0100] Embodiment 25. The compound of SEQ ID NO: 16, or a pharmaceutically acceptable salt thereof, wherein the compound comprises one or more protecting groups.

[0101] Embodiment 26. The compound of Embodiment 25, wherein the one or more protecting groups are selected from Fmoc, Boc, tert-butyl and trityl groups.

[0102] Embodiment 27. The compound of Embodiment 25 or 26, wherein the compound does not comprise one or more protecting groups of SEQ ID NO: 16 (ie, one or more protecting groups are removed from SEQ ID NO: 16).

[0103] Embodiment 28. The compound of Embodiment 25 or 26, wherein the compound does not comprise any protecting groups of SEQ ID NO: 16 (ie, all protecting groups are removed from SEQ ID NO: 16).

[0104] Embodiment 29. Method for preparing compound of formula (I):

[0105]

[0106] wherein PG is a protecting group, the method comprising:

[0107] (a) contacting the compound of formula (Ia) with diisopropylethylamine

[0108]

[0109] (b) contacting the product of step (a) with 2-(5-norbornene-2,3-dicarboximido)-1,1,3,3-tetramethyluronium tetrafluoroborate in DMF; and

[0110] (c) adding the compound of formula (Ib) to the mixture of (b),

[0111]

[0112] Embodiment 30. The method of Embodiment 29, wherein the protecting group is selected from Boc and Fmoc.

[0113] Embodiment 31. The method of Embodiment 29 or 30, wherein the protecting group is Fmoc.

[0114] Embodiment 32. The method of any one of Embodiments 29 to 31, wherein the method is performed using continuous flow.

[0115] Embodiment 33. The method of any one of Embodiments 29 to 32, wherein the method is performed in a flow reactor.

[0116] Embodiment 34. The method of any one of embodiments 1-12, wherein the method comprises filtering teilpotide or a pharmaceutically acceptable salt thereof under one or more of the following conditions: (i) a temperature of 10 to 34°C, optionally about 20°C; (ii) a turbulent / cross flow velocity of 1.94 to 2.46 m / s, optionally about 2.2 m / s; (iii) a laminar flow / shear rate of 2.1 x 10 3 to 2.7x10 3 1 / s, optionally about 2.4x10 3 1 / s; (iv) a primary product concentration of 32.8 to 47.2 mg / mL, optionally about 40 mg / mL; and (v) a secondary product concentration of 37 to 53 mg / mL, optionally about 45 mg / mL. Example

[0117] Example 1

[0118] Method 1: Synthesis of side chains using LPPS technology (SC100)

[0119]

[0120] Eicosanedioic acid mono(1,1-dimethylethyl) ester (15.0 kg, limiting reagent) and N-hydroxysuccinimide (1.2 eq) were dissolved in ethyl acetate at 27 ° C. A solution of DCC (1.25 eq) in ethyl acetate was added, and the reaction was stirred at 22 ° C for 24 hours. The resulting DCU by-product was filtered off, and the organic phase was extracted three times with 5% NaCl aqueous solution. After extraction, the organic phase was concentrated, co-evaporated with isopropanol, and then crystallized by adding heptane. After filtration, the filter cake was rinsed with heptane and dried at 25 ° C to obtain 17.0 kg of INT1 with a yield of 87% and a purity of 99%.

[0121] H-Glu-OtBu (7.7 kg, 1.1 equivalents) was dissolved in DCM (54 L) at 20 ° C, and then a solution of TMSA (11.3 kg) dissolved in DCM (7 L) was added, and the reaction mixture was stirred at 40 ° C for 1 hour. A DCM solution of INT1 (17.0 kg) was added at room temperature and stirred for 8 hours. After the reaction was completed, DCM was replaced with ethyl acetate by distillation. The organic phase was washed three times with a 2% KHSO4 / NaCl aqueous solution and then washed four times with a 2% NaCl aqueous solution. After separating and removing the aqueous phase, the organic phase was concentrated with isopropanol, diluted with isopropanol, and then crystallized by adding water. After filtration, the filter cake was washed with a water / isopropanol mixture and then dried at 30 ° C to obtain 17.3 kg of INT2 with a yield of 86% and a purity of 99%.

[0122] INT2 (17.3 kg) and N-hydroxysuccinimide (4.1 kg, 1.2 equiv) were dissolved in ethyl acetate (336 kg) at 27 ° C. A solution of DCC (8.33 kg, 1.25 equiv) in ethyl acetate was added, and the reaction was stirred at 22 ° C for 24 hours. The obtained DCU by-product was filtered off. The organic phase was concentrated, co-evaporated with isopropanol, and then crystallized by cooling the isopropanol solution (~125 L). Afterwards, the filter cake was rinsed with cold isopropanol and dried at 25 ° C to obtain 16.3 kg of INT3 with a yield of 81% and a purity of 96%.

[0123] 17-Amino-10-oxo-3,6,12,15-tetraoxa-9-azaheptadecanoic acid (AEEA2) (8.1 kg, 26.3 mol) was suspended in DCM (54 L) at 22 ° C, a solution of TMSA (7.68 kg, 59.9 mol) in DCM (6.2 L) was added, and the reaction mixture was stirred at 40 ° C for 1 hour. INT3 (16 kg) was suspended in DCM (31 L) at 35 ° C and then added to the TMS-protected (AEEA2) mixture at 22 ° C. The reactants were stirred for 12 hours. After the reaction was complete, the mixture was concentrated and then replaced with ethyl acetate. The organic phase was washed three times with a 2% KHSO4 / NaCl aqueous solution (~200 L) and then washed 4 times with a 2% NaCl aqueous solution (~200 L) until the target pH reached 4.5. The organic phase was concentrated and replaced with acetonitrile. The acetonitrile solution was cooled to -20 ° C and the resulting suspension was aged at -20 ° C for 15 hours. The mixture was filtered and the filter cake was rinsed with cold acetonitrile and then dried at <0°C to give 18.4 kg of SC100 (88% yield) with a purity of 96%. The overall yield was 53%. Method 2: The side chain SC100 was synthesized using a peptide synthesizer (SPPS technology).

[0124] Alternatively, when only an amide coupling reaction is desired, SC100 can be prepared using a peptide synthesizer using standard coupling procedures.

[0125] Standard coupling conditions included 0.133 M, 2.0 equivalents of HATU, and 5.0 equivalents of DIEA at ambient temperature for 3 hours, followed by deprotection using 3 x 15 min, 20% piperidine / DMF. The resin charge consisted of FmocNH-AEEA on 2-CTC resin (0.99 mmol / g), with 1.01 g used in each parallel reaction. An automated program was used, including a DMF swell followed by the addition of Pip / DMF; a DMF wash; and a mixture of amino acids, DIEA, and HATU; and a DMF wash cycle followed by drying. The combined batch was stirred in 30% HFIP / DCM (240 mL) for 1.5 hours to cleave the resin. The resin was filtered, washed, and the solvent was removed from the filtrate in vacuo. The resulting oil was dissolved in acetonitrile and the solvent was removed again. This operation yielded 30.47 g (146% theoretical yield) of a viscous yellow oil, which contained 52.3 area % of the desired product by UPLC analysis. The crude product was purified by flash chromatography (500 g silica gel, eluted with 85% DCM / 10% methanol / 5% acetic acid, collecting 38 x 100 mL fractions). The concentrate (17.94 g) from the previous chromatography purification was crystallized to give 13.4 g (74.7% yield) with a UPLC purity of 91.65 area%.

[0126] Method 3 (via Route 1 or Route 2): Side chain SC100 synthesis:

[0127]

[0128] Route 1:

[0129] To a mixture of dichloromethane, INT1 and N-hydroxysuccinimide (HOSu), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) are added over 1.5 to 3 hours. The reactants are stirred at 15°C to 25°C for 2 to 5 hours. Additional HOSu (target 0.09 equivalents) and EDCI (target 0.09 equivalents) may be added, and if the reaction is not complete, the reactants are stirred for an additional 2 to 5 hours. After the reaction is complete, the reactants are concentrated by distillation to remove dichloromethane. Thereafter, two acetonitrile addition cycles are performed, followed by a concentration step. After the second concentration, water is added to precipitate the product. The slurry is aged at 20°C to 30°C for 8 to 16 hours, then filtered and washed with a mixture of water and acetonitrile in a ratio of 2:1. The wet cake is transferred to a reactor and acetonitrile is added. The reactor is heated to 25°C to 35°C for 1 to 2 hours. The temperature was then lowered to -5°C to 5°C over 6 to 7 hours and aged for 30 minutes to 1 hour. The precipitated slurry was thermally cycled between -5°C to 5°C and 15°C to 25°C. Filtration was performed at -5°C to 5°C, and the wet cake was washed with cold acetonitrile. Thereafter, the product was dried under vacuum at 25°C to 35°C.

[0130] Step 2: A mixture of 17-amino-10-oxo-3,6,12,15-tetraoxa-9-azaheptadecanoic acid (AEEA2), dichloromethane and N-(trimethylsilyl)acetamide (TMSA) is stirred at 35°C to 45°C for 2 to 4 hours. Thereafter, the temperature is adjusted to 30°C to 35°C and INT2 is added. The reactants are stirred at 30°C to 35°C for 12 to 48 hours. After the reaction is complete, the mixture is cooled to 10°C to 20°C and washed with two portions of 3% KHSO4 and 25% NaCl in water, followed by one portion of 25% NaCl in water. After removing the aqueous layer, the organic layer is concentrated by distillation to remove dichloromethane. Thereafter, two acetonitrile addition cycles are performed, followed by a concentration step. The solution is then cooled to -25°C to -20°C to precipitate the product. The slurry was aged for 10 to 16 hours, filtered, washed with cold acetonitrile, and dried under vacuum at -20°C for 2 to 6 hours. The filter cake was then warmed to -15°C to -5°C and dried under vacuum for 6 to 10 hours, then warmed to -5°C to 3°C and continued to dry under vacuum. After drying was complete, DMF was added to dissolve the solid product to obtain a 20 to 30% w / w solution of SC100 in DMF.

[0131] Route 2:

[0132] Step 1: Add a mixture of acetonitrile, 4-dimethylaminopyridine (DMAP), and INT1 to N,N'-disuccinimidyl carbonate (DSC) at a target temperature of 35°C for a target reaction time of 3 hours. After the reaction is complete, cool the solution to -10°C to precipitate the product. The slurry is thermally cycled between -10°C and 20°C. After the temperature cycle is complete, filter the slurry and dry it at 25°C.

[0133] Step 2: A mixture of acetonitrile (ACN), 17-amino-10-oxo-3,6,12,15-tetraoxa-9-azaheptadecanoic acid (AEEA2) and N-methyl-N-trimethylsilyl acetamide (N-Me-N-TMSA) was stirred at a target temperature setting point of 25°C for 2 hours. Thereafter, INT2 was added to the mixture and maintained at a target temperature setting point of 25°C for 4 hours. After the reaction was complete, ethyl acetate (EtOAc) was added to the mixture, followed by washing with two portions of 2% potassium bisulfate / 1% NaCl water washes and two portions of 2% NaCl water washes. After the aqueous layer of the reaction solution was removed, EtOAc was removed by distillation. After removing EtOAc, ACN was added and removed by distillation four times. The solution was then cooled to a target temperature of -20°C to effect precipitation. The slurry was aged for 15 hours, and the product was filtered and washed with cold ACN. The solid was first dried under vacuum at -19°C to -13°C, then the temperature was raised to -10°C and finally lowered to -3°C to further dry the product. After drying was complete, DMF was added to dissolve the solid product to obtain a 20 to 30% w / w solution of SC100 in DMF.

[0134] Step 3 (Route 1 and Route 2):

[0135]

[0136] Step 3: A solution of SC100 in DMF was combined with diisopropylethylamine (DIPEA) in a flow reactor. The solution was passed through an in-line mixer and mixed with a solution of TNTU in DMF. After a target residence time of thirty (30) minutes, the resulting activated ester intermediate was mixed in-line with a solution of (((9H-fluoren-9-yl)methoxy)carbonyl)-L-lysine hydrochloride (Fmoc-Lys-OH.HCl) in DMF. After a target residence time of fifteen (15) minutes, the reaction was complete and 2-methyltetrahydrofuran (Me-THF) and sodium chloride / potassium bisulfate aqueous solution (NaCl / KHSO4 aqueous solution) were mixed in-line to quench the reaction and form a biphasic mixture. A batch extractive work-up was performed using NaCl / KHSO4 aqueous solution, methyl-THF, and DMF, followed by azeotropic distillation to remove Me-THF and water. Finally, DMF was added to obtain a 20-30% w / w solution of SC101 in DMF. SC101 is one of the compounds of formula (I) disclosed herein.

[0137] Example 2

[0138] Synthesis of Fmoc-hydrazine-CTC resin (Preparation Example 1)

[0139]

[0140] 2-CTC resin (10.7g, 17.7mmol) is swollen 20min at 0 DEG C in 100mL DCM. 9-fluorenylmethyl carbazate (9-fluorenylmethyl carbazate) (15.6g, 61.4mmol, 3.5 equivalents) is dissolved in 210mL 2:1DMF:DCM. DIEA (31mL, 178mmol, 10.1 equivalents) is added to 9-fluorenylmethyl carbazate solution. The solution is then slowly added to the resin at 0 DEG C. Stir for about 1 hour at 0 DEG C, then warm to room temperature. The reaction mixture is stirred at room temperature for 16 hours. Methanol (10mL) is then added to quench the remaining 2-CTC resin, and stir for 15min. The resin was rinsed with 200 mL of DMF, followed by DMF (2 x 100 mL), water (3 x 100 mL), DMF (3 x 100 mL), methanol (3 x 100 mL), and finally DCM (3 x 100 mL). The resin was dried in a vacuum oven at 27° C. for 16 hours. The resin loading was determined to be 0.74 mmol / g by quantitative NMR.

[0141] Example 3

[0142] Synthesis of peptide hydrazide fragment 1-17 (SEQ ID NO: 2)

[0143]

[0144] Hydrazine-CTC resin (1.01 g, loading: 0.65 mmol / g) was placed in a 40 mL reaction vessel and swollen on a peptide synthesizer with 3 x 4 mL of DCM (30 s each) followed by 2 x 10 mL of DMF (20 min each). Fmoc-Ile-OH (0.919 g, 2.60 mmol, 4 eq) and HBTU (0.99 g, 2.61 mmol, 4 eq) were dissolved in 7 mL of DMF. DIPEA (0.91 mL, 5.22 mmol, 8 eq) was added to the amino acid solution, and the volume was made up to 10 mL with DMF. The activated amino acid solution was added to the resin. The slurry was mixed with nitrogen for 8 hours. After 8 hours, the resin was washed with 5 x 10 mL of DMF and 5 x 10 mL of DCM and dried for 12 hours. The resulting resin loading was 0.54 mmol / g as measured by quantitative NMR. 0.91 g of the resin was used to synthesize peptide hydrazide fragment 1-17 (SEQ ID NO: 2).

[0145] Deprotection was performed using 4 x 9 mL of 20% v / v piperidine in DMF for 30 minutes each.

[0146] Amino acid coupling was performed using 3 equivalents of amino acid, 3 equivalents of OXYMA, and 3.3 equivalents of DIC. After each coupling and final iteration of deprotection, the resin was washed with 5 × 9 mL of DMF and mixed with N2 for 1 min. At the end of the peptide hydrazide synthesis, the resin was washed with DCM and mixed with N2. The resin was dried on the synthesizer.

[0147] Deprotection and cleavage: 25 mL of a cleavage mixture consisting of 5% w / v dithiothreitol (DTT), 2.5% v / v water, 2.5% v / v triisopropylsilane (TIPS), and 90% trifluoroacetic acid (TFA) was added to the dried resin (2.37 g) and mixed on a rotary mixer for 3 hours. The resin was filtered and washed with 2×2.5 mL TFA. The filtrate was poured into 175 mL cold MTBE, and the peptide immediately precipitated. The filter flask was washed with 2×2.0 mL TFA and poured into cold MTBE. The mixture was cooled to -20°C for half an hour and then centrifuged. The peptide precipitate was then washed twice with 150 mL MTBE and centrifuged. The peptide precipitate was dried in a vacuum oven at 27°C for 16 hours. After drying, a 1.25 g sample of crude peptide hydrazide fragment 1-17 (SEQ ID NO: 2) was obtained [expected value (mass + 2H +) / 2=968.4883,observed value(mass+2H + ) / 2=968.4879].

[0148] Example 4

[0149] Synthesis of Cysteine-18 fragment 18-39, linked to SC100 (SEQ ID NO: 3)

[0150]

[0151] Approximately 0.62 mmol of SEQ ID NO: 3 was synthesized on Sieber amide resin by standard SPPS protocol. Orthogonal deprotection and lysine acylation were performed using Fmoc-Lys(ivDde)-OH.

[0152] ivDde deprotection: Hydrazine hydrate (64% w / w) (1.98 g, 25.3 mmol) was diluted to 24.4 g with DMF, and 20 g was added to the resin. After approximately two hours, the slurry was stirred with a stream of nitrogen and washed with 5 x 9 mL of DMF. This step was repeated once.

[0153] 2-[2-[2-[[2-[2-[2-[[(4S)-5-tert-butoxy-4-[(20-tert-butoxy-20-oxo-eicosanoyl)amino]-5-oxo-pentanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetic acid (1094.4 mg, 1.252 mmol, 2 eq) was dissolved in 10 mL of anhydrous DMF. TNTU (506.9 mg, 1.360 mmol, 2.2 eq) and DIEA (0.24 mL, 1.4 mmol, 2.2 eq) were added. The volume was made up to 15 mL with anhydrous DMF. The mixture was mixed on a rotary mixer for 30 min. The activated ester of SC100 was then added to the resin and mixed with a stream of nitrogen for 12 hours. After 12 hours, the solution was drained and the resin was washed with 5 x 10 mL of DMF and 7 x 10 mL of DCM, mixing with N2 for 1 minute. The resin was dried on the synthesizer for 8 hours.

[0154] Deprotection and cleavage: 20 mL of a cleavage mixture consisting of 5% w / v dithiothreitol (DTT), 2.5% v / v water, 2.5% v / v triisopropylsilane (TIPS), and 90% trifluoroacetic acid (TFA) was added to a dried resin (2.42 g) and mixed on a rotary mixer for 3 hours. The resin was filtered and washed with 2 x 2.0 mL TFA. The filtrate was poured into 200 mL of cold MTBE, and the peptide precipitated immediately. The filter flask was washed with 2 x 2 mL TFA and then poured into cold MTBE. The precipitate was cooled to -20°C and held for 30 minutes, then centrifuged. The peptide precipitate was washed twice with 240 mL of MTBE and centrifuged. The peptide precipitate was dried in a vacuum oven at 27°C for 14 hours. After drying, 1.853 g of crude SEQ ID NO: 3 was obtained. It was purified by RP-HPLC on a Kromasil 100-10-C8 10 μm column (30 mm × 250 mm) at ambient temperature using a linear gradient of 15% acetonitrile in water for the first 5 min, followed by a linear gradient of 30% to 55% acetonitrile in water over 25 min, and finally eluted with a constant 0.1% TFA over 30 min. 1.28 g of purified SEQ ID NO: 3 [expected value (mass + 2H + ) / 2=1470.7929, measured value (mass+2H + ) / 2=1470.7885].

[0155] Example 5

[0156] Synthesis of Thioester Fragment 1-17 (SEQ ID NO: 4) (Converted from SEQ ID NO: 2)

[0157]

[0158] The crude hydrazide fragment 1-17 (SEQ ID NO: 2), 2.422 g, 1.251 mmol) was dissolved in 50 mL of ligation buffer (6 M guanidine hydrochloride and 0.2 M sodium hydrogen phosphate monobasic, pH 3.35) and cooled to -15°C in an acetone-ice bath. 9.4 mL of 1 M sodium nitrite solution (9.4 mmol, 7.5 equivalents) was added to the peptide hydrazide solution and stirred at -15°C for 20 min. Simultaneously, 1 mL of 2,2,2-trifluoroethanethiol (TFET) was added to 10 mL of ligation buffer (6 M guanidine hydrochloride and 0.2 M sodium hydrogen phosphate monobasic, pH 7.0). After 20 min, 10 mL of the TFET mixture was added to the peptide hydrazide solution to induce in situ thiolysis of the peptidyl azide generated by fragment 1-17 (SEQ ID NO: 2).

[0159] The pH of the reaction mixture was adjusted to about 6.95 with 5N sodium hydroxide solution. The thiolysis of the peptidyl azide was continued for 45 min, and the volume was made up to 100 mL with ligation buffer (pH 7.0). The crude thioester mixture was purified by RP-HPLC on a Waters X-Bridge C18 10 μm column (10 mm × 250 mm) at ambient temperature using a linear gradient of 10% acetonitrile in water for the first 2.8 min, followed by a linear gradient of 25% to 42% acetonitrile in water over 25 min, and finally eluted with a constant 0.1% TFA over 28 min. 1.03 g of TFET thioester SEQ ID NO: 4 [expected value (mass + 2H + ) / 2=1010.4650, measured value (mass+2H + ) / 2=1010.4620].

[0160] Example 6

[0161] The thioester fragment 1-17 (SEQ ID NO: 4) was reacted with the cysteine-18 fragment 18-39 (SEQ ID NO: 3) to form a thioester fragment. Then chemically linked to form the telpotide cysteine-18 analog (SEQ ID NO: 5):

[0162]

[0163] An aqueous solution of 6M guanidine hydrochloride and 0.3M sodium dihydrogen phosphate (pH 7.0) is the connection buffer for native chemical connection. All solutions are prepared in this connection buffer. 350.4mg (0.174mmol) peptide thioester SEQ ID NO:4 is dissolved in 50mL connection buffer. 8.0mL 0.5M 4-mercaptophenyl acetic acid (MPAA) solution is added to the peptide thioester solution. The N-terminal cysteine-containing peptide SEQ ID NO:3 (524.6mg, 0.178mmol, 1.03 equivalents) is dissolved in 48mL connection buffer in a 50mL centrifuge tube. SEQ ID NO:3 solution is added to the thioester solution. The tube is rinsed with 2.8mL connection buffer (about pH 7.0) and then added to the reaction mixture. The pH of the reaction mixture is adjusted to about 7 with 5N NaOH solution. 8.0 mL of tris(2-carboxyethyl)phosphine (TCEP, 0.5 M, pH 7.0) was added to the reaction mixture and the pH was adjusted to 7.0 again with 0.2 mL of 5N sodium hydroxide solution. The reaction was stirred at room temperature for 24 hours and then stored in a refrigerator. An additional 3 mL of 0.5 M TCEP solution was added and then purified. SEQ ID NO: 5 was purified by RP-HPLC on a Kromasil C18 10 μm column (10 mm × 250 mm) at ambient temperature. During the 28 min purification period, the first 4 min were eluted with a linear gradient of 10% acetonitrile in water, followed by a linear gradient elution from 20% to 50% acetonitrile in water over 23 min. After purification, approximately 372 mg (44.3%) of the teipotide cysteine analog SEQ ID NO: 5 [expected value (mass + 3H + ) / 3=1615.17263, measured value (mass+3H + ) / 3=1615.1686].

[0164] Desulfurization:

[0165]

[0166] An aqueous solution of 6-guanidine hydrochloride and 0.3M sodium dihydrogen phosphate (pH 7.0) is the buffer for desulfurization. All solutions are prepared in this buffer. 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (preparation 2,808.2 mg, 2.5 mmol) was dissolved in 10 mL of buffer, and the pH was adjusted to about 7.0 with 5N NaOH. The volume was made up to 15 mL with buffer. Telportide cysteine analog SEQ ID NO: 5 (105.2 mg, 0.022 mmol) was dissolved in 30 mL of buffer, and 6 mL of preparation example 2 solution was added thereto. 5 mL of 0.3 M L-glutathione reducing solution (L-GSH, pH 7.0) and 7.5 mL of 0.5 M TCEP solution (pH 7.0) were added thereto. The solution was heated at 44 ° C for 4.5 hours, and the reaction was found to be complete by UPLC analysis [expected value (mass+3H + ) / 3=1604.5153, measured value (mass+3H + ) / 3=1604.5122]. The desulfurization yield was calculated by UPLC using a telpotide (SEQ ID NO: 1) reference standard. The yield was estimated to be 47%.

[0167] Example 7

[0168] Synthesis of hydrazide fragment 1-20: SEQ ID NO: 6

[0169]

[0170] Hydrazine-CTC resin (2.03 g, 1.32 mmol, loading value: 0.65 mmol / g) was placed in a 40 mL reaction vessel and swelled with 3×10 mL DCM (30 s each) and then swelled with 2×10 mL DMF (20 min each) on a Symphony synthesizer. HBTU (1.48 g, 3.90 mmol, 3.0 equiv) was dissolved in 13.1 mL of a solution of (25S,52S)-52-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-25-(tert-butoxycarbonyl)-2,2-dimethyl-4,23,28,37,46-pentaoxo-3,32,35,41,44-pentaoxa-24,29,38,47-tetraazatripentacontan-53-oic acid (Preparation 3, 365 mg / mL in DMF) (3.91 mmol, 3.0 equiv). DIPEA (1.4 mL, 8.04 mmol, 6.1 equiv) was added to the solution, and the volume was made up to 19 mL with DMF. The solution was mixed at room temperature on a rotary mixer for 30 min. The activated ester solution from Preparation 3 was added to the resin. The slurry was mixed with nitrogen for 8 hours. After 8 hours, the resin was washed with 5×10 mL of DMF and 5×10 mL of DCM and dried for 12 hours. The loading of the resulting resin was determined to be 0.26 mmol / g by quantitative NMR. 1.82 g of this resin was used for the synthesis of peptide hydrazide (SEQ ID NO: 6).

[0171] Deprotection was performed using 4 x 9 mL of 20% v / v piperidine in DMF for 30 minutes each.

[0172] Coupling: Amino acid coupling was performed using 3 equivalents of amino acid, 3 equivalents of OXYMA and 3.3 equivalents of DIC.

[0173] After each coupling and the final deprotection repetition, the resin was washed with 5 × 9 mL of DMF and mixed with N2 for 1 min. At the end of the peptide hydrazide synthesis, the resin was washed with 7 × 10 mL of DCM and mixed with N2 for 1 min. The resin was then dried on the synthesizer for approximately 12 hours.

[0174] Deprotection and cleavage: 25 mL of a cleavage mixture consisting of 5% w / v dithiothreitol (DTT), 2.5% v / v water, 2.5% v / v triisopropylsilane (TIPS), and 90% trifluoroacetic acid (TFA) was added to the dried resin and mixed on a rotary mixer. The resin was filtered, washed with TFA (2 x 2.5 mL), and the filtrate was poured into 175 mL of cold MTBE. The filter flask was washed with TFA (2 x 2.5 mL), and the washings were poured into cold MTBE. The mixture was cooled to -20°C and held for 30 min, then centrifuged. The peptide precipitate was then washed twice with 150 mL of MTBE and centrifuged. The peptide precipitate was dried in a vacuum oven at 27°C for 16 hours. After drying, 1.70 g of crude peptide hydrazide SEQ ID NO: 6 was obtained. The crude peptide hydrazide SEQ ID NO: 6 was purified by RP-HPLC on a Waters XSelect CSH C18 10 μm column (10 mm × 250 mm) at ambient temperature using a linear gradient of 10% acetonitrile in water for the first 3 min, followed by a linear gradient of 20% to 55% acetonitrile in water over 23 min, and finally eluted with a constant 0.1% TFA over 28 min. Approximately 110 mg of partially purified hydrazide SEQ ID NO: 6 was obtained.

[0175] Deprotection and Cleavage: 25 mL of a cleavage mixture consisting of 5% w / v dithiothreitol (DTT), 2.5% v / v water, 2.5% v / v triisopropylsilane (TIPS), and 90% trifluoroacetic acid (TFA) was added to the dried resin (2.92 g) and mixed on a rotary mixer. The resin was filtered and washed with 2 x 2.5 mL TFA. The filtrate was poured into 200 mL of cold MTBE, and the peptide immediately precipitated. The filter flask was then washed with 2 x 2 mL TFA, and the washes were poured into cold MTBE. The mixture was cooled to –20°C for 30 min and then centrifuged. The peptide precipitate was then washed twice with 240 mL MTBE and centrifuged. The peptide precipitate was then dried in a vacuum oven at 27°C for 16 hours. 1.7 g of crude 19-mer SEQ ID NO: 8 was obtained.

[0176] Example 8

[0177] Native chemical ligation of fragment 1-20 (SEQ ID NO: 7) to cysteine fragment 21-39 (SEQ ID NO: 8) to form telpotide cysteine-21 analog (SEQ ID NO: 9)

[0178]

[0179] An aqueous solution of 6M guanidine hydrochloride and 0.3M sodium dihydrogen phosphate (pH 7.0) is the connection buffer for native chemical connection. All solutions are prepared in this connection buffer. Partially purified peptide hydrazide (SEQ ID NO: 6, 56 mg, 0.019 mmol) is dissolved in 5 mL of connection buffer (6M guanidine hydrochloride and 0.3M sodium dihydrogen phosphate, pH 3.35) and cooled to -15 ° C in an acetone-ice bath. 0.25 mL of 1M sodium nitrite solution (0.25 mmol, 13.2 equivalents) is added to the peptide hydrazide solution and stirred at -15 ° C for 10 min. After 10 min, 0.8 mL of 0.5M 4-mercaptophenylacetic acid (MPAA) solution is added to the peptide hydrazide solution to cause in situ thiolysis of the peptidyl azide generated by SEQ ID NO: 6. The pH of the reaction mixture is adjusted to about 7.0 with 5N sodium hydroxide solution. The thiolysis of the peptidyl azide is continued for 30 min.

[0180] Using a standard SPPS protocol, approximately 0.62 mmol of cysteine peptide 21-39 (SEQ ID NO: 8) was synthesized on a Sieber amide resin. The N-terminal cysteine containing SEQ ID NO: 8 (26.1 mg, 0.014 mmol, 0.74 equivalents) was dissolved in 1 mL of ligation buffer. The SEQ ID NO: 8 solution was added to the thioester solution. The vial containing SEQ ID NO: 8 was rinsed with 1 mL of ligation buffer (pH 7.0) and added to the reaction mixture. After 15 min, 1.0 mL of tris(2-carboxyethyl)phosphine (TCEP, 0.5 M, pH 7.0) was added to the reaction mixture, and the pH was adjusted to 7.0 with 5 N sodium hydroxide solution. The reactants were stirred at room temperature for 1 hour. Telportide cysteine analog (SEQ ID NO: 9) was observed in the reaction mixture.

[0181] Example 9

[0182] Native chemical ligation: protected fragment 1-21 (SEQ ID NO: 10) to protected fragment 22-39 (SEQ ID NO: 11)

[0183]

[0184] Reagent / Substrate Preparation: A 5 wt% solution of protected fragment 22-39 (SEQ ID NO: 11) (0.04 mmol, 1 eq, 95.9065 mg, 95.0 wt%) was prepared in DMF (1822.214 uL). Similarly, a 5 wt% solution of protected fragment 1-21 (SEQ ID NO: 10) (0.052 mmol, 1.38 eq, 268.077 mg, 79 wt%) was prepared in DMF (5093.46 uL). A 10% (v / v) solution of DIEA (0.084 mmol, 4.2 eq, 28.64 μL) was prepared in DMF (257.76 uL). A 10 wt% solution of HATU (0.04 mmol, 2.0 eq, 30.418 mg) was prepared in acetonitrile (273.762 uL).

[0185] Coupling and separation: Protected fragment 22-39 (SEQ ID NO: 11) and protected fragment 1-21 (SEQ ID NO: 10) were mixed together at 0°C. DIEA solution was added at 0°C, followed by HATU solution. The reaction was stirred at 0°C for 2 hours. 2.0 mL of 17% NaCl / 0.5% was added at 0°C, followed by 2.2 mL of cold water at 0°C and stirred for 1 hour. The off-white precipitate was filtered and washed with water. It was dried overnight in a vacuum oven at 40°C under an N2 atmosphere.

[0186] Global deprotection: The crude API isolated above was added to DCM (1.5 mL / g; 315.75 μL), 20 volumes of 92.25% TFA (3.87 mL), 2.5% DTT (0.105 mg), 2.5% water (0.105 mL), and 2.5% TIS (0.105 mL). Stir at room temperature for 2 h. Pour into cold MTBE (30 mL) and maintain at 2-4°C for 30 min. Then centrifuge at 3000 rpm for 3 min. The solid precipitate was washed with 2 x 30 mL of MTBE, centrifuging after each wash. Dry in vacuo at 40°C with a N2 purge. 159.13 mg of a white to off-white powder was obtained.

[0187] Example 10

[0188] Native chemical ligation: protected fragment 1-17 (SEQ ID NO: 13) to protected fragment 18-39 (SEQ ID NO: 14):

[0189]

[0190] Reagent / Substrate Preparation: A 5 wt% solution of protected fragment 18-39 (SEQ ID NO: 14) (0.02 mmol, 103.275 mg, 1 eq, 73.1 wt%) was weighed and dissolved in 1.96 mL of DMF. Similarly, a 5 wt% solution of protected fragment 1-17 (SEQ ID NO: 13) (0.02 mmol, 65.68 mg, 1.0 eq, 88.6 wt%) was weighed and dissolved in 1.24 mL of DMF. A 10% (v / v) solution of DIEA (0.084 mmol, 14.32 μL, 4.2 eq) in DMF (128.88 μL) was prepared. A 10 wt% solution of HATU (0.042 mmol, 2.0 eq, 15.96 mg) was prepared in 143.64 μL of acetonitrile.

[0191] Coupling and separation: Protected fragment 18-39 (SEQ ID NO: 14) and protected fragment 1-17 (SEQ ID NO: 13) were mixed together at 0°C. DIEA and HATU solutions were added to the mixture at 0°C. The reaction was stirred at 0°C for 2 hours. 2.0 mL of 17% NaCl / 0.5% was added at 0°C, followed by 2.2 mL of cold water at 0°C and stirred for 1 hour. The off-white precipitate was filtered and washed with water. It was dried overnight in a vacuum oven at 40°C under an N2 atmosphere.

[0192] Overall cleavage: The crude API isolated above was added to DCM (1.5 mL / g; 315.75 μL), 20 volumes of 92.25% TFA (3.87 mL), 2.5% DTT (0.105 mg), 2.5% water (0.105 mL), and 2.5% TIS (0.105 mL). Stirred at room temperature for 2 h. Poured into cold MTBE (30 mL) and kept at 2-4°C for 30 min. Then centrifuged at 3000 rpm for 3 min. The solid precipitate was washed with 2×30 mL MTBE, centrifuged after each wash. Drying was carried out at 40°C under vacuum with N2 purge. 159.13 mg of fully deprotected TZP (SEQ ID NO: 1) was obtained as an off-white powder.

[0193] Example 11

[0194] Native chemical ligation: Fragment 1-15 (SEQ ID NO: 15) to Fragment 16-39 (SEQ ID NO: 16):

[0195]

[0196]

[0197] Reagent / Substrate Preparation: A 5 wt% solution of protected fragment 16-39 (SEQ ID NO: 16) (0.02 mmol, 1 eq, 113.606 mg, 72.31 wt%) was prepared in DMF (2158.5 uL). Similarly, a 5 wt% solution of protected fragment 1-15 (SEQ ID NO: 15) (0.026 mmol, 1.30 eq, 66.95 mg, 88.76 wt%) was prepared in DMF (1272.05 uL). A 10% (v / v) solution of DIEA (0.084 mmol, 4.2 eq, 14.67 μL) was prepared in DMF (132 uL). A 10 wt% solution of HATU (0.042 mmol, 2.1 eq, 15.209 mg) was prepared in acetonitrile (143.64 uL).

[0198] Coupling and separation: Protected fragment 16-39 (SEQ ID NO: 16) and protected fragment 1-15 (SEQ ID NO: 15) were mixed together at 0°C. DIEA solution was added at 0°C, followed by HATU solution. The reaction was stirred at 0°C for 2 h. 2.0 mL of 17% NaCl / 0.5% was added at 0°C, followed by cold water (2.2 mL) at 0°C and stirred for 1 hour. The off-white precipitate was filtered and washed with water. It was dried overnight in a vacuum oven at 40°C under an N2 atmosphere.

[0199] Overall cleavage: The crude API isolated above was added to DCM (1.5 mL / g; 315.75 uL), 20 volumes of 92.25% TFA (3.87 mL), 2.5% DTT (0.105 mg), 2.5% water (0.105 mL), and 2.5% TIS (0.105 mL). It was stirred at room temperature for 2 h. Poured into cold MTBE (30 mL) and kept at 2-4°C for 30 min. It was then centrifuged at 3000 rpm for 3 minutes. The solid precipitate was washed with 2×30 mL MTBE, centrifuging after each wash. It was dried at 40°C under vacuum with N2 purge. 159.13 mg of a white to off-white powder was obtained.

[0200] Example 12: Amidation

[0201]

[0202] Fmoc-GPS(tBu)S(tBu)GAPPPS(tBu)-OH(SEQ ID NO:18) (1.0625 g, 1 equivalent) was added to a reaction vessel under an inert atmosphere and dissolved in 2-MeTHF (3.09 mL). The reaction vessel was placed in an ice bath and N-methylmorpholine (93.9 uL, 1 equivalent) was added to the solution. An additional 1 mL of 2-MeTHF was added. Isobutyl chloroformate (0.112 mL, 1 equivalent) was added to the reaction mixture, and the mixture was stirred for 10 minutes, followed by the addition of ammonium hydroxide (0.14 mL, 4 equivalents). The resulting mixture was warmed to room temperature and then mixed overnight (~18 hours). Equal amounts of EtOAc and water were then added to the reaction mixture. The aqueous layer was separated, and the organic layer was concentrated under reduced pressure to give Fmoc-GPS(tBu)S(tBu)GAPPPS(tBu)-NH2(SEQ ID NO:17) as a white solid. Measured Mass: 1242.6859 [M+H].

[0203] Example 13: Fmoc-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-OH (SEQ ID NO: 19)

[0204]

[0205] Crystallization screening of the free form of Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) was performed using a variety of solvents and solvent mixtures. Different crystallization methods were used, including solvent-based techniques such as slurry, cooling, ambient and sub-ambient temperatures holding, solvent / anti-solvent addition, or a combination of these techniques, as well as non-solvent-based techniques such as thermal stress.

[0206] Three solid forms were identified: Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form A, Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form C, and Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form D. Form A is a solvated form produced by 1-propanol (1-PrOH) or a mixture containing 1-PrOH (such as 1-PrOH / heptane). Form D is a solvated form produced by acetonitrile (ACN) or a mixture containing ACN (such as ACN / MTBE). Form C is a conversion product when Form D is isolated and dried.

[0207] Fmoc-GPS(tBu)-S(tBu)-G-OH(SEQID NO:19)Form A

[0208] Preparation Example 1:

[0209] Fmoc-GPS(tBu)-S(tBu)-G-OH(SEQ ID NO:19) Form A was prepared in 1-propanol (1-PrOH). 16 mL of 1-PrOH was added to 4.06 grams of Fmoc-GPS(tBu)-S(tBu)-G-OH(SEQ ID NO:19) amorphous solid, and the sample was stirred at ambient conditions to obtain an orange-red solution. 2.8 mg of Fmoc-GPS(tBu)-S(tBu)-G-OH(SEQ ID NO:19) Form A was added to the solution, and stirring was continued for 2 days at ambient conditions. A light orange suspension was obtained, and the solid was isolated by vacuum filtration using a 10 μm disposable filter, rinsed twice on the filter with w / 0.5 mL of fresh 1-PrOH, then collected and dried at 30°C under vacuum for about 3-4 hours. The resulting white solid (3.2 g) was Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form A.

[0210] Preparation Example 2:

[0211] Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form A was prepared in 1-propanol (1-PrOH) and heptane. Approximately 50 mg of amorphous Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) was dissolved in 0.6 mL of 1-PrOH to form a clear yellow solution. A 30 μL aliquot of the solution was added to 0.6 mL of 1-PrOH to form a clear light yellow solution. With stirring, an additional 0.6 mL of heptane was added to the solution, the sample was covered, and stirred at ambient conditions for 2 days. A suspension was obtained, and the resulting solid was Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form A.

[0212] Preparation Example 3:

[0213] Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form A was prepared in 1-propanol (1-PrOH). Approximately 10 mg of amorphous Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) was dissolved in 0.1 mL of 1-PrOH to form a clear yellow solution. The solution was stored in a capped vial under ambient conditions for 1 day and then transferred to a refrigerator for 3 days. The solid observed in the solution was Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form A.

[0214] XRPD of Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form A

[0215] XRPD pattern of crystalline Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form A was obtained by XRPD analysis using a CuKα The X-ray powder diffractometer was obtained on a Bruker D8 Endeavor X-ray powder diffractometer equipped with a source and a Linxeye detector, operating at 40 kV and 40 mA. The sample was scanned between 4 and 42 2θ° with a step size of 0.009 2θ° and a scan rate of 0.5 s / step, using a 0.3° primary slit opening and a 3.9° PSD opening. The powder was loaded on a quartz sample holder and a glass slide was used to obtain a smooth surface. The diffraction patterns were collected at ambient temperature and relative humidity. The crystal peak positions were determined in MDI-Jade v7.9.9.

[0216] The prepared Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form A sample was characterized by an XRPD pattern using CuKα radiation, and its diffraction peaks (2θ values) are shown in Table 1 below, including a peak at 6.1°2θ in combination with one or more peaks selected from 8.5, 11.7, 12.3, and 16.9°2θ; the diffraction angle tolerance is 0.2 degrees. A representative XRPD pattern of Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form A is shown in Table 1 below. Figure 1A shown.

[0217] Table 1. X-ray powder diffraction peaks of Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form A

[0218]

[0219] Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form C

[0220] Preparation Example 1:

[0221] 0.5 mL of ACN was added to 66.9 mg of Fmoc-GPS(tBu)-S(tBu)-G-OH(SEQ ID NO: 19) Form A, and the sample was stirred at 51 ° C to obtain a clear colorless solution. The solution was moved to ambient conditions, and a small amount of Fmoc-GPS(tBu)-S(tBu)-G-OH(SEQ ID NO: 19) Form A solid was added to the sample. The resulting thin suspension was stirred for 3 days under ambient conditions. A white suspension was obtained, and the solid was separated using a 0.45 μm nylon syringe filter and centrifuged for 5 minutes under ambient conditions. The white solid on the filter was placed under ambient conditions and air-dried for about 20 minutes under a gentle stream of N2. The resulting white solid is Fmoc-GPS(tBu)-S(tBu)-G-OH(SEQ ID NO: 19) Form C.

[0222] Preparation Example 2:

[0223] 8Ml 2: 1v / v of ACN / MTBE was added to 2.25 grams of Fmoc-GPS (tBu) -S (tBu) -G-OH (SEQ ID NO: 19) Form A, and the sample was stirred at ambient conditions to obtain a white suspension. Fmoc-GPS (tBu) -S (tBu) -G-OH (SEQ ID NO: 19) Form C was added to the suspension, and then stirring was continued for 2 days at ambient conditions. The solid was separated by vacuum filtration using a 10 μm disposable filter, rinsed with w / 1 mL of fresh 2: 1 ACN / MTBE on the filter, and the solid was collected and dried under vacuum at 30 ° C. The dried white solid is Fmoc-GPS (tBu) -S (tBu) -G-OH Form C.

[0224] XRPD of Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form C

[0225] The XRPD pattern of crystalline Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form C was obtained using the same procedure as for Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form A.

[0226] The prepared Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form C sample was characterized by an XRPD pattern using CuKα radiation, and its diffraction peaks (2θ values) are shown in Table 2 below, including a peak at 6.1°2θ in combination with one or more peaks selected from 8.7, 10.6, 14.1, and 15.8°2θ; the tolerance for the diffraction angle is 0.2 degrees. A representative XRPD pattern of Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form C is shown in Table 2 below. Figure 1B shown.

[0227] Table 2. X-ray powder diffraction peaks of Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form C

[0228]

[0229] Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO:19) Form D

[0230] Preparation Example 1:

[0231] 1 mL of 1:1 v / v ACN / MTBE was added to 72.6 mg of Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form A, and the sample was stirred at ambient conditions to give a white suspension. Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form C was added to the suspension, and stirring was continued at ambient conditions for 2 days. The wet solid in the slurry was Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form D, which was physically unstable and converted to Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form C after isolation / drying.

[0232] XRPD of Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form D

[0233] The XRPD pattern of crystalline Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form C was obtained using the same procedure as for Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form A, except that the Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form B sample was scanned between 4 and 25 2θ° at a scan rate of 0.1 sec / step.

[0234] The prepared Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form D sample was characterized by an XRPD pattern using CuKα radiation, and its diffraction peaks (2θ values) are shown in Table 3 below, including a peak at 6.1°2θ in combination with one or more peaks selected from 4.1, 10.4, and 12.7°2θ; the tolerance for the diffraction angle is 0.2 degrees. A representative XRPD pattern of Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form D is shown in Table 3 below. Figure 1C shown.

[0235] Table 3. X-ray powder diffraction peaks of Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form D

[0236]

[0237] Example 14: Gelation Studies

[0238] The gelation properties of the peptide fragments were investigated under different conditions, including different process solvents (DMSO / ACN and DMF), peptide fragment concentration, gelation over time, processing shear rate (by varying the cross-flow velocity), temperature conditions, and gelation reversibility.

[0239] Process solvents

[0240] The gel properties of the peptide fragments in DMSO / ACN and DMF at different peptide fragment concentrations were studied. Figure 2A Viscosity measurements (Pa.s) at a shear rate of 1 / s are shown. The results show that the peptide product in DMF starts to become more viscous at lower concentration values compared to DMSO / CAN.

[0241] Concentration and time

[0242] The gel properties of the peptide fragments were studied in DMSO / ACN and DMF at different peptide fragment concentrations over 4 days. Figure 2B Viscosity measurements (Pa.s) at a shear rate of 1 / s are shown. The results show that in both solvent systems, the viscosity of the formulations increases with increasing peptide fragment concentration and also increases over time.

[0243] shear rate

[0244] The gel properties of the peptide fragments at different shear rate conditions were studied when the concentrations of the peptide fragments in DMF were 40 mg / mL, 50 mg / mL, 60 mg / mL and 70 mg / mL. Figure 2CViscosity measurements (Pa.s) are shown. The results show that the viscosity of the peptide fragments in DMF remains consistent with increasing shear rate at concentrations of 40 mg / mL and 50 mg / mL, while the viscosity of the peptide fragments in DMF decreases significantly with increasing shear rate at concentrations of 60 mg / mL and 70 mg / mL.

[0245] The gel properties of the peptide fragments at different shear rate conditions were studied when the concentrations of the peptide fragments were 50 mg / mL, 60 mg / mL and 70 mg / mL in DMSO / ACN. Figure 2D Viscosity measurements (Pa.s) are shown. The results show that the viscosity of the peptide fragment at 50 mg / mL and 60 mg / mL concentrations in DMSO / ACN remains consistent with increasing shear rate, while the viscosity of the peptide fragment at 70 mg / mL concentration in DMSO / ACN decreases significantly with increasing shear rate.

[0246] Parameter range

[0247] The results of the gelation study were used to study and determine improved parameter conditions (temperature, concentration, shear rate, cross flow velocity) for the nanofiltration of the peptide fragments disclosed herein. The improved parameter conditions included: (i) temperature from 10°C to 34°C (target temperature of approximately 20°C), (ii) turbulent flow / cross flow velocity from 1.94 to 2.46 m / s (target temperature of approximately 2.2 m / s); (iii) laminar flow / shear rate from 2.1×10 3 to 2.7×10 3 l / s(target temperature is about 2.4×10 3 l / s); (iv) primary concentration targets from 32.8 to 47.2 mg / mL (target temperature approximately 40 mg / mL); (v) secondary concentration targets from 37 to 53 mg / mL (target temperature approximately 45 mg / mL).

[0248] Viscosity can be reduced using high temperatures, low peptide fragment concentrations, high shear rates, and high cross-flow velocities to reduce viscosity and thereby reverse gelation.

[0249] sequence

[0250] SEQ ID NO:1–Telbotepeptide

[0251]

[0252] where K is replaced at position 20 by (2-[2-(2-aminoethoxy)-ethoxy]-acetyl)2-(γGlu)1-CO-(CH2) 18 -CO2H was coupled to the ε-amino group of the K side chain for chemical modification.

[0253] SEQ ID NO: 2-Hydrazide fragment 1-17

[0254]

[0255] SEQ ID NO: 3-Cysteine-18 fragment 18-39

[0256]

[0257] SEQ ID NO: 4-thioester fragment 1-17

[0258]

[0259] SEQ ID NO: 5-TZP cysteine-18 analog

[0260]

[0261] SEQ ID NO: 6-Hydrazide fragment 1-20

[0262]

[0263] SEQ ID NO: 7-thioester fragment 1-20

[0264]

[0265] SEQ ID NO: 8-Cysteine-21 fragment 21-39

[0266]

[0267] SEQ ID NO:9-TZP cysteine-21 analog

[0268] SEQ ID NO: 10 - Protected fragments 1-21

[0269]

[0270] SEQ ID NO: 11 - Protected fragments 22-39

[0271]

[0272] SEQ ID NO: 12 - Protected TZP

[0273]

[0274] SEQ ID NO: 13 - Protected fragments 1-17

[0275]

[0276] SEQ ID NO: 14 - Protected fragments 18-39

[0277]

[0278] SEQ ID NO: 15 - Protected fragments 1-15

[0279]

[0280] SEQ ID NO: 16 - Protected fragment 16-39

[0281]

Claims

1. A method for preparing tilpotide or a pharmaceutically acceptable salt thereof, comprising: (a) coupling the peptide of SEQ ID NO: 10 with SEQ ID NO: 11 to form the peptide of SEQ ID NO: 12; as well as (b) deprotecting the peptide of SEQ ID NO: 12 to obtain telpotide or a pharmaceutically acceptable salt thereof.

2. The method of claim 1, wherein the peptide of SEQ ID NO: 12 from step (a) is isolated and then subjected to a deprotection step (b).

3. The method of claim 1 or 2, wherein the peptide of SEQ ID NO: 12 from step (a) is washed prior to the deprotection step (b).

4. The method of claim 1 or 2, wherein after the deprotection step (b), the peptide of SEQ ID NO: 12 from step (a) is washed.

5. A method for preparing tilpotide or a pharmaceutically acceptable salt thereof, comprising: (a) coupling the peptide of SEQ ID NO: 13 with SEQ ID NO: 14 to form the peptide of SEQ ID NO: 12; as well as (b) deprotecting the peptide of SEQ ID NO: 12 to obtain telpotide or a pharmaceutically acceptable salt thereof.

6. The method of claim 5, wherein the peptide of SEQ ID NO: 12 is isolated from step (a) prior to step (b).

7. The method of claim 5 or 6, wherein the peptide of SEQ ID NO: 12 from step (a) is washed prior to the deprotection step (b).

8. The method of claim 5 or 6, wherein after the deprotection step (b), the peptide of SEQ ID NO: 12 from step (a) is washed.

9. A method for preparing tilpotide or a pharmaceutically acceptable salt thereof, the method comprising: (a) coupling the peptide of SEQ ID NO: 15 to SEQ ID NO: 16 to form the peptide of SEQ ID NO: 12; as well as (b) deprotecting the peptide of SEQ ID NO: 12 to obtain telpotide or a pharmaceutically acceptable salt thereof.

10. The method of claim 9, wherein the peptide of SEQ ID NO: 12 is isolated from step (a) prior to step (b).

11. The method of claim 9 or 10, wherein the peptide of SEQ ID NO: 12 from step (a) is washed prior to the deprotection step (b).

12. The method of claim 9 or 10, wherein after the deprotection step (b), the peptide of SEQ ID NO: 12 from step (a) is washed.

13. The compound of SEQ ID NO: 10 or a pharmaceutically acceptable salt thereof, wherein the compound comprises one or more protecting groups.

14. The compound of claim 13, wherein the one or more protecting groups are selected from the group consisting of Fmoc, Boc, tert-butyl, and trityl.

15. The compound of claim 13 or 14, wherein the compound does not comprise one or more protecting groups of SEQ ID NO:

10.

16. The compound of claim 13 or 14, wherein the compound does not comprise any protecting groups of SEQ ID NO:

10.

17. The compound of SEQ ID NO: 13 or a pharmaceutically acceptable salt thereof, wherein the compound comprises one or more protecting groups.

18. The compound of claim 17, wherein the one or more protecting groups are selected from the group consisting of Fmoc, Boc, tert-butyl, and trityl.

19. The compound of SEQ ID NO: 14 or a pharmaceutically acceptable salt thereof, wherein the compound comprises one or more protecting groups.

20. The compound of claim 19, wherein the one or more protecting groups are selected from the group consisting of Fmoc, Boc, tert-butyl, and trityl.

21. The compound of SEQ ID NO: 15 or a pharmaceutically acceptable salt thereof, wherein the compound comprises one or more protecting groups.

22. The compound of claim 21, wherein the one or more protecting groups are selected from Fmoc, Boc, tert-butyl and trityl groups.

23. The compound of claim 21 or 22, wherein the compound does not comprise one or more protecting groups of SEQ ID NO:

15.

24. The compound of claim 21 or 22, wherein the compound does not comprise any protecting groups of SEQ ID NO:

15.

25. The compound of SEQ ID NO: 16 or a pharmaceutically acceptable salt thereof, wherein the compound comprises one or more protecting groups.

26. The compound of claim 25, wherein the one or more protecting groups are selected from the group consisting of Fmoc, Boc, tert-butyl, and trityl.

27. The compound of claim 25 or 26, wherein the compound does not comprise one or more protecting groups of SEQ ID NO:

16.

28. The compound of claim 25 or 26, wherein the compound does not comprise any protecting groups of SEQ ID NO:

16.

29. A method for preparing a compound of formula (I): wherein PG is a protecting group, the method comprising: (a) contacting the compound of formula (Ia) with diisopropylethylamine, (b) contacting the product of step (a) with 2-(5-norbornene-2,3-dicarboximido)-1,1,3,3-tetramethyluronium tetrafluoroborate in DMF; and (c) adding the compound of formula (Ib) to the mixture of (b), 30. The method of claim 29, wherein the protecting group is selected from the group consisting of Boc and Fmoc.

31. The method of claim 29 or 30, wherein the protecting group is Fmoc.

32. The method of any one of claims 29 to 31 , wherein the method is performed using continuous flow.

33. The process of any one of claims 29 to 32, wherein the process is performed in a flow reactor.

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