Process and intermediates for preparing tirzepatide

Abstract

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

Description

[Technical Field]

[0001] (Related Applications) This application claims priority to U.S. Provisional Patent Application No. 63 / 477,734, filed December 29, 2022, the entire contents of which are incorporated herein by reference.

[0002] (Sequence Listing) This application contains a Sequence Listing that has been submitted electronically in XML format, and is incorporated herein by reference in its entirety. The XML copy created on December 27, 2023 is named 02021-0166-00304_SL.XML and is 78,828 bytes in size.

[0003] FIELD OF THE INVENTION The present disclosure provides processes and intermediates for making a GIP / GLP1 dual agonist peptide (referred to herein as "tirzepatide") or a pharmaceutically acceptable salt thereof. [Background technology]

[0004] Diabetes is a chronic disease characterized by hyperglycemia due to defects in insulin secretion, insulin action, or both. In type 2 diabetes mellitus ("T2D"), the combined effects of impaired insulin secretion and insulin resistance are associated with elevated blood glucose levels. Tirzepatide ("TZP"), a GIP / GLP1 dual agonist, is described and claimed in U.S. Patent No. 9,474,780. Tirzepatide may be useful in the treatment of T2D. U.S. Patent No. 9,474,780 is incorporated herein by reference in its entirety.

[0005] Large-scale preparation of pharmaceutically acceptable tirzepatide presents several technical challenges that can affect overall yield and purity. Processes and intermediates that enable improved or alternative manufacturing methods for tirzepatide are needed. Similarly, efficient, environmentally "friendly" processes, including stable intermediates for producing tirzepatide with fewer purification steps, are needed. Improved or alternative technologies are also needed to provide tirzepatide manufacturing processes that minimize waste streams to enhance both environmental and operator safety. Processes are also needed to avoid the use of transition metals and / or harsh reaction conditions that are incompatible with peptide synthesis.

[0006] The present disclosure seeks to meet these needs by providing novel intermediates and processes useful for the production of tirzepatide (SEQ ID NO: 1), or a pharmaceutically acceptable salt thereof. The disclosed tirzepatide production process provides intermediates and process reactions that embody a combination of advances, including an efficient route with fewer steps, while maintaining high quality and purity. In addition, the new process and intermediates reduce resource intensity and minimize waste streams.

[0007] The novel process described herein provides various embodiments of intermediates useful in the preparation of tirzepetide. Summary of the Invention [Means for solving the problem]

[0008] The present disclosure describes methods for preparing tirzepatide or a pharmaceutically acceptable salt thereof.

[0009] In certain embodiments, the present disclosure describes a method for preparing tirzepatide or a pharmaceutically acceptable salt thereof, the method comprising: (a) coupling a peptide of SEQ ID NO: 10 to 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 tirzepatide or a pharmaceutically acceptable salt thereof. In certain embodiments, the peptide of SEQ ID NO: 12 from step (a) is isolated before the deprotection step (b). In certain embodiments, the peptide of SEQ ID NO: 12 from step (a) is washed before the deprotection step (b). In certain embodiments, the peptide of SEQ ID NO: 12 from step (a) is washed after the deprotection step (b).

[0010] In certain embodiments, the present disclosure describes a method for preparing tirzepatide or a pharmaceutically acceptable salt thereof, the method comprising: (a) coupling a peptide of SEQ ID NO: 13 to 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 tirzepatide or a pharmaceutically acceptable salt thereof. In certain embodiments, the peptide of SEQ ID NO: 12 from step (a) is isolated before step (b). In certain embodiments, the peptide of SEQ ID NO: 12 from step (a) is washed before the deprotection step (b). In certain embodiments, the peptide of SEQ ID NO: 12 from step (a) is washed after the deprotection step (b).

[0011] In certain embodiments, the present disclosure describes a method for preparing tirzepatide or a pharmaceutically acceptable salt thereof, the method comprising: (a) coupling a peptide of SEQ ID NO: 15 to 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 tirzepatide or a pharmaceutically acceptable salt thereof. In certain embodiments, the peptide of SEQ ID NO: 12 from step (a) is isolated before step (b). In certain embodiments, the peptide of SEQ ID NO: 12 from step (a) is washed before the deprotection step (b). In certain embodiments, the peptide of SEQ ID NO: 12 from step (a) is washed after the deprotection step (b).

[0012] 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 includes one or more protecting groups. In certain embodiments, the one or more protecting groups are selected from Fmoc, Boc, tert-butyl, and trityl groups. In certain embodiments, the compound does not include one or more of the 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 include any of the protecting groups of SEQ ID NO: 10 (i.e., all protecting groups are removed from SEQ ID NO: 10).

[0013] In certain embodiments, the disclosure describes a compound of SEQ ID NO: 13, or a pharmaceutically acceptable salt thereof. In certain embodiments, the disclosure describes a compound of SEQ ID NO: 13, or a pharmaceutically acceptable salt thereof, wherein the compound includes one or more protecting groups. In certain embodiments, the one or more protecting groups are selected from Fmoc, Boc, tert-butyl, and trityl groups.

[0014] In certain embodiments, the disclosure describes a compound of SEQ ID NO: 14, or a pharmaceutically acceptable salt thereof. In certain embodiments, the disclosure describes a compound of SEQ ID NO: 14, or a pharmaceutically acceptable salt thereof, wherein the compound includes one or more protecting groups. In certain embodiments, the one or more protecting groups are selected from Fmoc, Boc, tert-butyl, and trityl groups.

[0015] In certain embodiments, the present disclosure describes a compound of SEQ ID NO: 15, or a pharmaceutically acceptable salt thereof. In certain embodiments, the present disclosure describes a compound of SEQ ID NO: 15, or a pharmaceutically acceptable salt thereof, wherein the compound includes one or more protecting groups. In certain embodiments, the one or more protecting groups are selected from Fmoc, Boc, tert-butyl, and trityl groups. In certain embodiments, the compound does not include one or more of the protecting groups of SEQ ID NO: 15 (i.e., one or more protecting groups are removed from SEQ ID NO: 15). In certain embodiments, the compound does not include any of the protecting groups of SEQ ID NO: 15 (i.e., all protecting groups are removed from SEQ ID NO: 15).

[0016] In certain embodiments, the present disclosure describes a compound of SEQ ID NO: 16, or a pharmaceutically acceptable salt thereof. In certain embodiments, the present disclosure describes a compound of SEQ ID NO: 16, or a pharmaceutically acceptable salt thereof, wherein the compound includes one or more protecting groups. In certain embodiments, the one or more protecting groups are selected from Fmoc, Boc, tert-butyl, and trityl groups. In certain embodiments, the compound does not include one or more of the protecting groups of SEQ ID NO: 16 (i.e., one or more protecting groups are removed from SEQ ID NO: 16). In certain embodiments, the compound does not include any of the protecting groups of SEQ ID NO: 16 (i.e., all protecting groups are removed from SEQ ID NO: 16).

[0017] In certain embodiments, the present disclosure describes a process for preparing a compound of formula (I):

[0018] [ka] wherein PG is a protecting group and the process comprises: (a) contacting a compound of Formula (Ia) with diisopropylethylamine;

[0019] [ka] (b) contacting the product of step (a) with 2-(5-norbornene-2,3-dicarboximido)-1,1,3,3-tetramethyluronium tetrafluoroborate in DMF; (c) adding a compound of formula (Ib) to the mixture of (b).

[0020] [ka]

[0021] In certain embodiments, the protecting group is selected from Boc and Fmoc. In certain embodiments, the protecting group is Fmoc. In certain embodiments, the process is carried out using continuous flow. In certain embodiments, the process is carried out in a flow reactor.

[0022] In certain embodiments, the present disclosure describes a method comprising filtering tirzepatide 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 turbulence / cross-flow velocity of 1.94 to 2.46 m / s, optionally about 2.2 m / s; (iii) a flow rate of 2.1 x 10 3 ~2.7×10 3 1 / s, optionally approximately 2.4 × 10 3 a laminar flow / shear rate of 1 / s, (iv) a primary product concentration of 32.8-47.2 mg / mL, optionally about 40 mg / mL, and (v) a secondary product concentration of 37-53 mg / mL, optionally about 45 mg / mL. [Brief explanation of the drawings]

[0023] [Figure 1A] 1 shows representative XRPD patterns of Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) pentamer Form A, Form C, and Form D, respectively. [Figure 1B] 1 shows representative XRPD patterns of Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) pentamer Form A, Form C, and Form D, respectively. [Figure 1C]2A, 2B, 2C, and 2D show representative XRPD patterns of Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) pentamer Form A, Form C, and Form D, respectively. 2A, 2B, 2C, and 2D show viscosity measurements of peptide fragment gelation studies of the present disclosure. [Figure 2A] Figure 2A shows viscosity measurements of a peptide fragment gelation study of the present disclosure. Figure 2A shows viscosity measurements of a gelation study in relation to process solvent. [Figure 2B] Figure 2A shows viscosity measurements from the peptide fragment gelation study of the present disclosure. Figure 2B shows viscosity measurements from the gelation study in relation to peptide concentration and gelation time. [Figure 2C] Figure 2C shows viscosity measurements of the peptide fragment gelation study of the present disclosure. Figure 2C shows viscosity measurements of the gelation study in relation to peptide concentration and process shear rate. [Figure 2D] Figure 2D shows viscosity measurements of the peptide fragment gelation study of the present disclosure in relation to peptide concentration and process shear rate. DETAILED DESCRIPTION OF THE INVENTION

[0024] The present disclosure provides a method for preparing tirzepatide or a pharmaceutically acceptable salt thereof. In some embodiments, the method includes the step (a) of coupling a peptide of SEQ ID NO: 10 to a peptide of SEQ ID NO: 11 to form a peptide of SEQ ID NO: 12.

[0025] The present disclosure also provides a method for preparing tirzepatide or a pharmaceutically acceptable salt thereof, comprising step (a) coupling a peptide of SEQ ID NO: 13 to a peptide of SEQ ID NO: 14 to form a peptide of SEQ ID NO: 12.

[0026] The present disclosure also provides a method for preparing tirzepatide or a pharmaceutically acceptable salt thereof, comprising step (a) coupling a peptide of SEQ ID NO: 15 to a peptide of SEQ ID NO: 16 to form a peptide of SEQ ID NO: 12.

[0027] In some embodiments, the method further comprises the step (b) of deprotecting the peptide of SEQ ID NO: 12 to obtain tirzepatide or a pharmaceutically acceptable salt thereof.

[0028] 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 the deprotection step (b). In some embodiments, the method comprises washing the coupled product of step (a) after the deprotection step (b).

[0029] 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 includes one or more protecting groups.

[0030] 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 includes one or more protecting groups.

[0031] 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 includes one or more protecting groups.

[0032] 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 includes one or more protecting groups.

[0033] 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 includes one or more protecting groups.

[0034] In some embodiments, the one or more protecting groups are selected from Fmoc, Boc, tert-butyl, and trityl groups, 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 groups. In some embodiments, the compound is protected with an Fmoc group. In some embodiments, the compound is protected with Fmoc and Boc groups. In some embodiments, the compound is protected with Fmoc and tert-butyl groups. In some embodiments, the compound is protected with Fmoc and trityl groups. In some embodiments, the compound is protected with a Boc group. In some embodiments, the compound is protected with Boc and tert-butyl groups. In some embodiments, the compound is protected with Boc and trityl groups. In some embodiments, the compound is protected with a tert-butyl group. In some embodiments, the compound is protected with tert-butyl and trityl groups. In some embodiments, the compound is protected with a trityl group. In some embodiments, the compound is protected with Fmoc, Boc, and tert-butyl groups. In some embodiments, the compound is protected with Fmoc, Boc, and trityl groups. In some embodiments, the compound is protected with Boc, tert-butyl, and trityl groups. In some embodiments, the compound is protected with Fmoc, tert-butyl, and trityl groups.

[0035] In some embodiments, one or more protecting groups are removed in step (b). In some embodiments, all protecting groups are removed in step (b).

[0036] In another aspect of the present disclosure, there is provided a process for preparing tirzepatide side chain + lysine of formula (I):

[0037] [ka] In the formula, PG is a protecting group.

[0038] 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) does not comprise a protecting group. In some embodiments, the compound of formula (I) is used to prepare tirzepatide. In some embodiments, the compound of formula (I) is used to prepare tirzepatide using a linear SPPS method.

[0039] In some embodiments, the process for preparing a compound of Formula (I) comprises step (a) contacting a compound of Formula (Ia) with diisopropylethylamine;

[0040] [ka] In the formula, PG is a protecting group.

[0041] In some embodiments, the process for producing a compound of Formula (I) further comprises step (b) of 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 process comprises step (c) of adding a protected lysine of Formula (Ib) to the mixture of step (b).

[0042] [ka]

[0043] 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.

[0044] In some embodiments, the process for producing a compound of Formula (I) is carried out using a continuous flow reactor. In some embodiments, the process for producing a compound of Formula (I) is carried out in a flow reactor.

[0045] 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 fluorenylmethyloxycarbonyl chloride, "HATU" means (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, "HFIP" means hexafluoroisopropanol, and "IPA" means isopropyl alcohol. propanol, "L-GSH" means L-glutathione reducing solution, "LPPS" means liquid phase peptide synthesis, "MTBE" means methyl tert-butyl ether, "OXYMA" means ethyl cyanohydroxyiminoacetate, "Pip" means piperidine, "SPPS" means solid phase peptide synthesis, "TFA" means trifluoroacetic acid, "TFET" means 2,2,2-trifluoroethanethiol, "TIPS" means triisopropylsilane, "TCEP" means tris(2-carboxyethyl)phosphine, "TMSA" means trimethylsilylacetamide, "TNTU" means 2-(5-norbornene-2,3-dicarboximido)-1,1,3,3-tetramethyluronium tetrafluoroborate, "TZP" means tirzepatide, and "UPLC" means ultra-performance liquid chromatography.

[0046] As shown herein, single-letter amino acid abbreviations are shown in bold, and atoms are shown as non-bold text to distinguish them from single-letter amino acid abbreviations. As used herein, when an amino acid abbreviation appears with a number above the amino acid, that number refers to the position of the corresponding amino acid in the final tirzepatide product. Numbers are provided for convenience, and the presence or absence of such numbers in a sequence does not affect the amino acid sequence or peptide shown in such sequence.

[0047] As used herein, the term "protected" means that a protecting group is attached at the indicated position. Those skilled in the art will recognize that a variety of protecting groups are well known and that alternative protecting groups may be suitable for a particular process.

[0048] As used herein, the term "protecting group" or "amino acid protecting group" refers to a group that protects the acid or amine moiety of an amino acid or a reactive moiety on the side chain of an amino acid. An "acid moiety" includes, for example, a carboxylic acid group (-COOH). An "amine moiety" includes, for example, a primary amine group (-NH), a secondary amine group (-NH-), an amide group (-C(O)-NH), and a guanidinium group ([-NHC(NH)-NH]). + ). The acid or amine moiety can be part of the terminal amino acid in a peptide or polypeptide, or on the side chain of a non-terminal amino acid in a peptide or polypeptide. Other reactive moieties on the side chain of amino acids include, for example, hydroxy (-OH) and thiol (-SH) groups.

[0049] The protecting group may be a removable group known in the art to (i) protect a reactive group (such as an amine or carboxylic acid group) against undesired reactions during synthetic procedures, e.g., to block or protect the functionality of a reactive group during reactions involving other functional portions of a compound, and (ii) to be selectively deprotected in a multiply protected structure without affecting other protecting groups. Suitable protecting groups and methods for introducing and removing such groups include those known in the art, such as those described in T.W. Green and P.G.M. Buts, 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] Suitable protecting groups for lysine (Lys) include, but are not limited to, t-butoxycarbonyl (Boc), allyloxycarbonyl (Alloc), 4-phenylacetoxybenzyloxycarbonyl (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.

[0055] 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.

[0056] 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.

[0057] Exemplary acid protecting groups include esters, such as substituted and unsubstituted C1-C8 lower alkyl (e.g., methyl, ethyl, t-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, Examples of the alkyl group include alkoxycarbonyloxyalkyl (e.g., t-butyloxycarbonyloxymethyl), alkoxycarbonylaminoalkyl (e.g., t-butyloxycarbonylaminomethyl), alkylaminocarbonylaminoalkyl (e.g., methylaminocarbonylaminomethyl), acylaminoalkyl (e.g., acetylaminomethyl), heterocyclylcarbonyloxyalkyl (e.g., 4-methylpiperazinyl-carbonyloxymethyl), dialkylaminocarbonylalkyl (e.g., dimethylaminocarbonyl-methyl), (5-(lower alkyl)-2-oxo-1,3-dioxolen-4-yl)alkyl (e.g., (5-t-butyl-2-oxo-1,3-dioxolen-4-yl)methyl), and (5-phenyl-2-oxo-1,3-dioxolen-4-yl)alkyl (e.g., (5-phenyl-2-oxo-1,3-dioxolen-4-yl)methyl).

[0058] Exemplary amine and / or amide protecting groups include, but are not limited to, acyl (e.g., formyl, acetyl, chloroacetyl, trichloroacetyl, o-nitrophenylacetyl, o-nitrophenoxy-acetyl, trifluoroacetyl, acetoacetyl, 4-chlorobutyryl, isobutyryl, o-nitrocinnamoyl, picolinoyl, acylisothiocyanate, aminocaproyl, benzoyl), acyloxy (e.g., methoxy-carbonyl, 9-fluorenylmethoxycarbonyl, 2,2,2-trifluoroethoxycarbonyl, 2-trimethylsilylethoxy-carbonyl, vinyloxycarbonyl, allyloxycarbonyl, t-butyloxycarbonyl (Boc), 1,1-dimethyl-propynyloxycarbonyl, benzyloxycarbonyl (Cbz), p-nitrobenzyloxycarbonyl, 2,4-dichloro-benzyloxycarbonyl), 9-xanthenyl, and trityl. Further exemplary amide protecting groups include, but are not limited to, o-nitrocinnamoyl, picolinoyl, aminocaproyl, benzoyl, acyloxy (e.g., methoxycarbonyl, 9-fluorenylmethoxycarbonyl, 2,2,2-trifluoroethoxycarbonyl, 2-trimethylsilylethoxycarbonyl, vinyloxycarbonyl, allyloxycarbonyl, t-butyloxycarbonyl (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 t-butyloxycarbonyl (Boc). Exemplary imidazole protecting groups include, but are not limited to, tosyl (To), benzyloxymethyl (Bom), trityl (Trt), and t-butyloxycarbonyl (Boc). Exemplary guanidinium protecting groups include, but are not limited to, 2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl (Pbf) and t-butyloxycarbonyl (Boc).

[0059] Exemplary hydroxyl protecting groups include, but are not limited to, unsubstituted or substituted alkyl (e.g., t-butyl, allyl, benzyl, methoxymethyl, tetrahydropyranyl, o-nitrobenzyl), silyl (e.g., t-butyldimethylsilyl (TBDMS), t-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).

[0060] Those skilled in the art will appreciate that there are a variety of resins available for constructing the peptides presented herein. For example, those skilled in the art are familiar with Sieber and Rink amide resins for preparing the peptides disclosed herein; however, alternative resins may be selected for preparing the peptides described herein. For example, resins such as, but not limited to, 2-CTC and related resins may be used to prepare the target peptide, followed by a C-terminal amidation step.

[0061] The solid-phase peptide synthesis (SPPS) builds described herein are achieved using standard fluorenylmethyloxycarbonyl chloride (Fmoc) peptide chemistry techniques with sequential coupling on an automated peptide synthesizer. In some embodiments, the resin is swollen in DMF and then deprotected using 20% ​​piperidine (Pip) / DMF (3 x 30 min). In some embodiments, subsequent Fmoc deprotection involves 3 x 30 min treatments of 20% Pip / DMF, with 4 x 30 min treatments being used for more difficult couplings. In some embodiments, after deprotection, the resin is washed with 5 x 2 min washes of 10 volumes of DMF. In some embodiments, preactivation of amino acids involves diisopropylcarbodiimide (DIC) / ethyl cyanohydroxyiminoacetate (OXYMA) DMF solution for 30 min at room temperature. In some embodiments, coupling of activated amino acids to resin-bound peptides is performed for the time specified for each individual amino acid. In some embodiments, each coupling is followed by 5 x 2 min solvent washes with 10 volumes of DMF. In some embodiments, to isolate the final product, the resin-bound product is washed with 10 volumes of DCM for 5 × 2 minutes to remove DMF. In some embodiments, the resin is washed with 10 volumes of IPA for 2 × 2 minutes to remove DCM, and then washed with 10 volumes of methyl tert-butyl ether (MTBE) for 5 × 2 minutes, after which the product is dried under vacuum at 40 °C. In some embodiments, the resin-bound product is stored refrigerated (-20 °C). In some embodiments, for analysis, the peptide is cleaved from the resin using an acidic cocktail consisting of trifluoroacetic acid (TFA) / HO / TIPS (triisopropylsilane) / DTT (dithiothreitol) in the following ratio: 0.93 v / 0.04 v / 0.03 v / 0.03 w. In some embodiments, the resin is swelled with DCM (4–5 mL, 3 × 30 minutes) and allowed to drain. In some embodiments, the cleavage cocktail (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 then the resin is washed with a small amount of DCM and mixed with the cleavage solution.In some embodiments, the resulting solution is poured into 7-10 volumes of cold (0°C) methyl tert-butyl ether (MTBE). In some embodiments, the suspension is aged at 0°C for 30 minutes, then the resulting precipitate is 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 centrifuged and decanted again. In some embodiments, after decanting, the clear MTBE solution of the precipitated peptide is dried under vacuum at 40°C overnight.

[0062] As described in this disclosure, native chemical ligation is a useful process for preparing full-length peptides containing cysteine ​​or alanine in their sequence. This process utilizes the chemoselective reaction of two unprotected peptide segments to generate a temporary thioester-linked intermediate. The thioester-linked intermediate rearranges to provide a full-length ligation product with a native peptide bond at the ligation site. Those skilled in the art will appreciate that the technique of native chemical ligation can be useful for the chemical synthesis of full-length peptides containing cysteine ​​or alanine.

[0063] Enumerated Embodiments Embodiment 1. A method for preparing tirzepatide or a pharmaceutically acceptable salt thereof, comprising: (a) coupling a peptide of SEQ ID NO: 10 to 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 tirzepatide or a pharmaceutically acceptable salt thereof.

[0064] Embodiment 2. The method of embodiment 1, wherein the peptide of SEQ ID NO: 12 from step (a) is isolated prior to the deprotection step (b).

[0065] Embodiment 3. The method of embodiment 1 or 2, wherein the peptide of SEQ ID NO: 12 from step (a) is washed before the deprotection step (b).

[0066] Embodiment 4. The method of embodiment 1 or 2, wherein the peptide of SEQ ID NO: 12 from step (a) is washed after the deprotection step (b).

[0067] Embodiment 5. A method for preparing tirzepatide or a pharmaceutically acceptable salt thereof, comprising: (a) coupling a peptide of SEQ ID NO: 13 to 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 tirzepatide or a pharmaceutically acceptable salt thereof.

[0068] Embodiment 6. The method of embodiment 5, wherein the peptide of SEQ ID NO: 12 from step (a) is isolated prior to step (b).

[0069] Embodiment 7. The method of embodiment 5 or 6, wherein the peptide of SEQ ID NO: 12 from step (a) is washed before the deprotection step (b).

[0070] Embodiment 8. The method of embodiment 5 or 6, wherein the peptide of SEQ ID NO: 12 from step (a) is washed after the deprotection step (b).

[0071] Embodiment 9. A method for preparing tirzepatide or a pharmaceutically acceptable salt thereof, comprising: (a) coupling a peptide of SEQ ID NO: 15 to 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 tirzepatide or a pharmaceutically acceptable salt thereof.

[0072] Embodiment 10. The method of embodiment 9, wherein the peptide of SEQ ID NO: 12 from step (a) is isolated before step (b).

[0073] Embodiment 11. The method of embodiment 9 or 10, wherein the peptide of SEQ ID NO: 12 from step (a) is washed before the deprotection step (b).

[0074] Embodiment 12. The method of embodiment 9 or 10, wherein the peptide of SEQ ID NO: 12 from step (a) is washed after the deprotection step (b).

[0075] Embodiment 13. A compound of SEQ ID NO: 10, or a pharmaceutically acceptable salt thereof, comprising one or more protecting groups.

[0076] Embodiment 14. The compound of embodiment 13, wherein the one or more protecting groups are selected from Fmoc, Boc, tert-butyl, and trityl groups.

[0077] Embodiment 15. The compound of embodiment 13 or 14, wherein the compound does not include one or more of the protecting groups of SEQ ID NO: 10 (i.e., one or more protecting groups are removed from SEQ ID NO: 10).

[0078] Embodiment 16. The compound of embodiment 13 or 14, wherein the compound does not contain any of the protecting groups of SEQ ID NO: 10 (i.e., all protecting groups are removed from SEQ ID NO: 10).

[0079] Embodiment 17. A compound of SEQ ID NO: 13, or a pharmaceutically acceptable salt thereof, comprising one or more protecting groups.

[0080] Embodiment 18. The compound of embodiment 17, wherein the one or more protecting groups are selected from Fmoc, Boc, tert-butyl, and trityl groups.

[0081] Embodiment 19. A compound of SEQ ID NO: 14, or a pharmaceutically acceptable salt thereof, comprising one or more protecting groups.

[0082] Embodiment 20. The compound of embodiment 19, wherein the one or more protecting groups are selected from Fmoc, Boc, tert-butyl, and trityl groups.

[0083] Embodiment 21. A compound of SEQ ID NO: 15, or a pharmaceutically acceptable salt thereof, comprising one or more protecting groups.

[0084] Embodiment 22. The compound of embodiment 21, wherein the one or more protecting groups are selected from Fmoc, Boc, tert-butyl, and trityl groups.

[0085] Embodiment 23. The compound of embodiment 21 or 22, wherein the compound does not comprise one or more of the protecting groups of SEQ ID NO: 15 (i.e., one or more protecting groups are removed from SEQ ID NO: 15).

[0086] Embodiment 24. The compound of embodiment 21 or 22, wherein the compound does not contain any of the protecting groups of SEQ ID NO: 15 (i.e., all protecting groups are removed from SEQ ID NO: 15).

[0087] Embodiment 25. A compound of SEQ ID NO: 16, or a pharmaceutically acceptable salt thereof, comprising one or more protecting groups.

[0088] Embodiment 26. The compound of embodiment 25, wherein the one or more protecting groups are selected from Fmoc, Boc, tert-butyl, and trityl groups.

[0089] Embodiment 27. The compound of embodiment 25 or 26, wherein the compound does not comprise one or more of the protecting groups of SEQ ID NO: 16 (i.e., one or more protecting groups are removed from SEQ ID NO: 16).

[0090] Embodiment 28. The compound of embodiment 25 or 26, wherein the compound does not contain any of the protecting groups of SEQ ID NO: 16 (i.e., all protecting groups are removed from SEQ ID NO: 16).

[0091] Embodiment 29. A process for preparing a compound of formula (I), comprising:

[0092] [ka] wherein PG is a protecting group and the process is (a) contacting a compound of Formula (Ia) with diisopropylethylamine;

[0093] [ka] (b) contacting the product of step (a) with 2-(5-norbornene-2,3-dicarboximido)-1,1,3,3-tetramethyluronium tetrafluoroborate in DMF; (c) adding a compound of formula (Ib) to the mixture of (b);

[0094] [ka] The process includes:

[0095] Embodiment 30. The process of embodiment 29, wherein the protecting group is selected from Boc and Fmoc.

[0096] Embodiment 31 The process of embodiment 29 or 30, wherein the protecting group is Fmoc.

[0097] Embodiment 32. The process of any one of embodiments 29 to 31, wherein the process is carried out using continuous flow.

[0098] Embodiment 33. The process of any one of embodiments 29 to 32, wherein the process is carried out in a flow reactor.

[0099] Embodiment 34. The method of any one of embodiments 1 to 12, comprising filtering tirzepatide 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 turbulence / cross-flow velocity of 1.94 to 2.46 m / s, optionally about 2.2 m / s; (iii) a turbulence / cross-flow velocity of 2.1 x 10 3 ~2.7×10 3 1 / s, optionally approximately 2.4 × 10 3a laminar flow / shear rate of 1 / s, (iv) a primary product concentration of 32.8-47.2 mg / mL, optionally about 40 mg / mL, and (v) a secondary product concentration of 37-53 mg / mL, optionally about 45 mg / mL. [Example]

[0100] Example 1 Method 1: Side chain (SC100) synthesis using LPPS technology

[0101] [ka]

[0102] Eicosanedioic acid, mono(1,1-dimethylethyl) ester (15.0 kg, limiting reactant) and N-hydroxysuccinimide (1.2 equiv.) were dissolved in ethyl acetate at 27°C. A solution of DCC (1.25 equiv.) dissolved in ethyl acetate was added, and the reaction was stirred at 22°C for 24 hours. The resulting DCC by-product was removed by filtration, and the organic phase was extracted three times with 5% aqueous NaCl. After extraction, the organic phase was concentrated, coevaporated with isopropanol, and crystallized by adding heptane. After filtration, the filter cake was rinsed with heptane and dried at 25°C to give 17.0 kg of INT1 in 87% yield and 99% purity.

[0103] H-Glu-OtBu (7.7 kg, 1.1 equiv.) was dissolved in DCM (54 L) at 20 °C. 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 h. INT1 (17.0 kg) in DCM was added at room temperature and stirred for 8 h. After the reaction was complete, DCM was exchanged for ethyl acetate by distillation. The organic phase was washed three times with 2% KHSO4 / NaCl aqueous solution, then four times with 2% NaCl aqueous solution. After separating and removing the aqueous phase, the organic phase was concentrated with isopropanol, diluted with isopropanol, and crystallized by adding water. After filtration, the filter cake was washed with a water / isopropanol mixture and then dried at 30 °C to produce 17.3 kg of INT2 in 86% yield and 99% purity.

[0104] 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 resulting DCU by-product was filtered off. The organic phase was concentrated and coevaporated with isopropanol, followed by crystallization by cooling the isopropanol solution (approximately 125 L). The filter cake was then rinsed with cold isopropanol and dried at 25 °C to give 16.3 kg of INT3 in 81% yield and 96% purity.

[0105] 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 h. INT3 (16 kg) in DCM (31 L) was suspended at 35 °C and added to the TMS-protected (AEEA2) mixture at 22 °C. The reaction was stirred for 12 h. Upon completion, the mixture was concentrated and then exchanged into ethyl acetate. The organic phase was washed three times with 2% KHSO4 / NaCl aqueous solution (approximately 200 L), followed by four times with 2% NaCl aqueous solution (approximately 200 L) to a target pH of 4.5. The organic phase was concentrated and exchanged into acetonitrile. The acetonitrile solution was cooled to −20° C., and the resulting suspension was then 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%.

[0106] Method 2: Side chain SC100 synthesis using a peptide synthesizer (SPPS technology) Alternatively, SC100 can be prepared using a peptide synthesizer if only an amide coupling reaction is required, utilizing standard coupling procedures.

[0107] Standard coupling conditions included 0.133 M, 2.0 equiv. HATU and 5.0 equiv. DIEA for 3 h at ambient temperature, followed by deprotection using 20% ​​piperidine / DMF for 3 × 15 min. The resin load included FmocNH-AEEA (0.99 mmol / g) on ​​2-CTC resin, with 1.01 g used in each of parallel reactions. An automated program was used, including a DMF swell, followed by the addition of Pip / DMF, a DMF wash, and a combination of amino acid, DIEA, HATU, and a DMF wash cycle, followed by drying. The resin was cleaved by stirring the combined lots with 30% HFIP / DCM (240 mL) for 1.5 h. The resin was filtered and washed, and the solvent was removed from the filtrate under vacuum. The resulting oil was dissolved in acetonitrile, and the solvent was again removed. This procedure yielded 30.47 g (146% of theoretical yield) of a viscous yellow oil, containing 52.3 area % of the desired product by UPLC analysis. The crude product was purified by flash chromatography (500 grams of silica gel, eluting with 85% DCM / 10% methanol / 5% acetic acid, collecting 38 × 100 mL fractions). The previously chromatographed concentrate (17.94 g) was crystallized to give 13.4 g (74.7% yield) with a UPLC purity of 91.65 area %.

[0108] Method 3 (via Route 1 or Route 2): Side Chain SC100 Synthesis:

[0109] [ka]

[0110] Route 1: Step 1: To a mixture of dichloromethane, INT1, and N-hydroxysuccinimide (HOSu), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) was added over 1.5–3 hours. The reaction was stirred at 15–25°C for 2–5 hours. Additional HOSu (target 0.09 equivalents) and EDCI (target 0.09 equivalents) could be charged, and if the reaction was incomplete, the reaction could be stirred for an additional 2–5 hours. After the reaction was complete, the reaction was concentrated by distillation to remove the dichloromethane. This was followed by two cycles of adding acetonitrile and then concentrating. After the second concentration, water was added to precipitate the product. The slurry was aged at 20–30°C for 8–16 hours, then filtered and washed with a 2:1 mixture of water:acetonitrile. The wet cake was transferred to a reactor and charged with acetonitrile. The reactor was heated at 25–35°C for 1–2 hours. The temperature was then lowered to -5°C to 5°C over 6-7 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. Following this, the product was dried under vacuum at 25°C to 35°C.

[0111] Step 2: A mixture of 17-amino-10-oxo-3,6,12,15-tetraoxa-9-azaheptadecanoic acid (AEEA2), dichloromethane, and N-(trimethylsilyl)acetamide (TMSA) was stirred at 35°C to 45°C for 2 to 4 hours. Following this, the temperature was adjusted to 30°C to 35°C, and INT2 was added. The reaction was stirred at 30°C to 35°C for 12 to 48 hours. Upon completion of the reaction, the mixture was cooled to 10°C to 20°C and washed with two aqueous washes of 3% KHSO4 and 25% NaCl, followed by an aqueous wash of 25% NaCl. After removing the aqueous layer, the organic layer was concentrated by distillation to remove the dichloromethane. This was followed by two cycles of the addition of acetonitrile followed by concentration. The solution was then cooled to -25°C to -20°C to precipitate the product. The slurry was aged for 10-16 hours, filtered, washed with cold acetonitrile, and dried under vacuum at -20°C for 2-6 hours. The cake was then warmed to -15°C to -5°C and dried under vacuum for 6-10 hours, after which the cake was 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, yielding a 20-30% w / w SC100 solution in DMF.

[0112] Route 2: Step 1: To a mixture of acetonitrile, 4-dimethylaminopyridine (DMAP), and INT1, N,N'-disuccinimidyl carbonate (DSC) was added at a target temperature of 35°C for a target reaction time of 3 hours. After the reaction was complete, the solution was cooled to -10°C to precipitate the product. The slurry was thermocycled between -10°C and 20°C. After temperature cycling, the slurry was filtered and dried at 25°C.

[0113] Step 2: A mixture of acetonitrile (ACN), 17-amino-10-oxo-3,6,12,15-tetraoxa-9-azaheptadecanoic acid (AEEA2), and N-methyl-N-trimethylsilylacetamide (N-Me-N-TMSA) was stirred for 2 hours at a target temperature setpoint of 25 °C. Following this, INT2 was charged to the mixture, and the reaction was allowed to proceed for 4 hours at a target temperature setpoint of 25 °C. After the reaction was complete, ethyl acetate (EtOAc) was added to the mixture, followed by washing twice with 2% potassium bisulfate / 1% NaCl aqueous solution and twice with 2% NaCl aqueous solution. After removing the aqueous layer of the reaction mixture, the EtOAc was removed by distillation. After removing the EtOAc, ACN was charged and removed by distillation four times. The solution was then cooled to a target temperature of -20 °C to induce 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 increased to −10° C. and finally to −3° C. to further dry the product. After drying was complete, DMF was added to dissolve the solid product, yielding a 20–30% w / w SC100 solution in DMF.

[0114] Step 3 (Route 1 and Route 2):

[0115] [ka]

[0116] 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 combined with a solution of TNTU in DMF. After a target residence time of 30 min, 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 15 min, the reaction was complete and quenched by in-line mixing of 2-methyltetrahydrofuran (Me-THF) and aqueous sodium chloride / potassium hydrogen sulfate (aqueous NaCl / KHSO4), creating a biphasic mixture. A batch extractive workup using aqueous NaCl / KHSO4, Me-THF, and DMF was performed, followed by azeotropic distillation to remove Me-THF and water. Finally, DMF was charged to obtain a 20–30% w / w solution of SC101 in DMF. SC101 is one of the compounds of formula (I) described in the present disclosure.

[0117] Example 2 Synthesis of Fmoc-hydrazine-CTC resin (Preparation 1)

[0118] [ka]

[0119] 2-CTC resin (10.7 g, 17.7 mmol) was swollen in 100 mL of DCM at 0 °C for 20 min. 9-Fluorenylmethyl carbazate (15.6 g, 61.4 mmol, 3.5 equiv.) was dissolved in 210 mL of 2:1 DMF:DCM. DIEA (31 mL, 178 mmol, 10.1 equiv.) was added to the 9-fluorenylmethyl carbazate solution. This solution was then added slowly to the resin at 0 °C. The mixture was stirred at 0 °C for approximately 1 h and allowed to warm to room temperature. The reaction mixture was stirred at room temperature for 16 h. Methanol (10 mL) was then added to quench the remaining 2-CTC resin and stirred for 15 min. 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 h. The loading of the resin was determined to be 0.74 mmol / g by quantitative NMR.

[0120] Example 3 Synthesis of peptide hydrazide fragment 1-17: (SEQ ID NO: 2)

[0121] [ka]

[0122] Hydrazine-CTC resin (1.01 g, loading: 0.65 mmol / g) was placed in a 40 mL reaction vessel and swollen with 3 × 4 mL of DCM (30 s each), followed by 2 × 10 mL of DMF (20 min each) on a peptide synthesizer. Fmoc-Ile-OH (0.919 g, 2.60 mmol, 4 equiv.) and HBTU (0.99 g, 2.61 mmol, 4 equiv.) were dissolved in 7 mL of DMF. DIPEA (0.91 mL, 5.22 mmol, 8 equiv.) was added to the amino acid solution, and the volume was brought to 10 mL with DMF. The activated amino acid solution was added to the resin. The slurry was mixed with nitrogen for 8 h. After 8 h, the resin was washed with 5 × 10 mL of DMF and 5 × 10 mL of DCM and dried for 12 h. The loading of the resulting resin was determined to be 0.54 mmol / g by quantitative NMR. 0.91 g of this resin was used for the synthesis of peptide hydrazide fragment 1-17 (SEQ ID NO:2).

[0123] Deprotection was carried out using 4 x 9 mL of 20% v / v piperidine in DMF for 30 min each.

[0124] Amino acid couplings were performed using 3 equivalents of amino acid, 3 equivalents of OXYMA, and 3.3 equivalents of DIC. After each coupling and final deprotection iteration, the resin was washed with 5 x 9 mL of DMF for 1 minute with N2 mixing. After peptide hydrazide synthesis, the resin was washed with DCM with N2 mixing. The resin was allowed to dry on the synthesizer.

[0125] Deprotection and cleavage: 25 mL of cleavage cocktail made with 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 of TFA. The filtrate was poured into 175 mL of cold MTBE, and the peptide was immediately precipitated. The filter flask was washed with 2 × 2.0 mL of TFA and poured into cold MTBE. After cooling to -20 °C for 30 minutes, it was 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. A 1.25 g sample of crude peptide hydrazide fragment 1-17 (SEQ ID NO: 2) was obtained after drying [expected value (mass + 2H]]. + ) / 2=968.4883, observed value (mass + 2H + ) / 2=968.4879].

[0126] Example 4 Synthesis of SC100-linked cysteine-18 fragment 18-39: (SEQ ID NO: 3)

[0127] [ka]

[0128] Approximately 0.62 mmol of SEQ ID NO: 3 was synthesized on Sieber amide resin by standard SPPS protocols. Fmoc-Lys(ivDde)-OH was used for orthogonal deprotection and lysine acylation.

[0129] Deprotection of ivDde: Hydrazine monohydrate (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. The slurry was stirred with a stream of nitrogen and after approximately 2 hours was washed with 5 x 9 mL of DMF. Repeat once more.

[0130] 2-[2-[2-[[2-[2-[2-[[(4S)-5-tert-butoxy-4-[(20-tert-butoxy-20-oxo-icosanoyl)amino]-5-oxo-pentanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetic acid (1094.4 mg, 1.252 mmol, 2 equiv.) was dissolved in 10 mL of anhydrous DMF. TNTU (506.9 mg, 1.360 mmol, 2.2 equiv.) and DIEA (0.24 mL, 1.4 mmol, 2.2 equiv.) were added. The volume was brought to 15 mL with anhydrous DMF. Mixing was allowed to continue on a rotary mixer for 30 minutes. The activated ester of SC100 was then added to the resin and mixed with a nitrogen stream 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 for 1 minute with N mixing. The resin was dried on the synthesizer for 8 hours.

[0131] Deprotection and cleavage: 20 mL of cleavage cocktail made with 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 (2.42 g) and mixed on a rotary mixer for 3 hours. The resin was filtered and washed with 2 × 2.0 mL of TFA. The filtrate was poured into 200 mL of cold MTBE, and the peptide was immediately precipitated. The filter flask was washed with 2 × 2 mL of TFA and poured into cold MTBE. After cooling to -20 °C for 30 minutes, it was 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. Purification was performed by RP-HPLC on a Kromasil 100-10-C8 10 μm column (30 mm × 250 mm) at ambient temperature. After an initial 5 min of 15% acetonitrile in water, a linear gradient of 30-55% acetonitrile in water over 25 min, followed by a constant 0.1% TFA gradient over 30 min, was used. 1.28 g of purified SEQ ID NO: 3 was obtained [expected (mass + 2H + ) / 2=1470.7929, observed value (mass + 2H + ) / 2=1470.7885].

[0132] Example 5 Synthesis of thioester fragment 1-17 (SEQ ID NO:4) (from conversion of SEQ ID NO:2)

[0133] [ka]

[0134] 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 disodium 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 equiv.) was added to the peptide hydrazide solution and stirred at -15 °C for 20 min. Meanwhile, 1 mL of 2,2,2-trifluoroethanethiol (TFET) was added to the peptide hydrazide solution (6 M guanidine hydrochloride and 0.2 M disodium hydrogen phosphate monobasic, pH 7.0) to bring the volume up to 10 mL. After 20 min, 10 mL of the TFET mixture was added to the peptide hydrazide solution to allow in situ thiolytic cleavage of the peptidyl azide generated from fragment 1-17 (SEQ ID NO:2).

[0135] The pH of the reaction mixture was adjusted to approximately 6.95 with 5N sodium hydroxide solution. Thiol cleavage of the peptidyl azide was carried out for 45 min, and the volume was brought 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. After an initial 2.8 min of 10% acetonitrile in water, a linear gradient of 25 to 42% acetonitrile in water over 25 min, with 0.1% TFA constant throughout the 28 min purification, was used. This yielded 1.03 g of TFET thioester SEQ ID NO: 4 [expected (mass + 2H]). + ) / 2=1010.4650, observed value (mass + 2H + ) / 2=1010.4620].

[0136] Example 6 Native chemical ligation of thioester fragment 1-17 (SEQ ID NO: 4) to cysteine-18 fragment 18-39 (SEQ ID NO: 3) to form the tirzepatide cysteine-18 analog (SEQ ID NO: 5):

[0137] [ka]

[0138] An aqueous solution of 6 M guanidine hydrochloride and 0.3 M disodium hydrogen phosphate monobasic (pH 7.0) was used as the ligation buffer for native chemical ligation. All solutions were prepared with this ligation buffer. 350.4 mg (0.174 mmol) of peptide thioester SEQ ID NO:4 was dissolved in 50 mL of ligation buffer. 8.0 mL of 0.5 M 4-mercaptophenylacetic acid (MPAA) solution was added to the peptide thioester solution. The N-terminal cysteine-containing peptide SEQ ID NO:3 (524.6 mg, 0.178 mmol, 1.03 equiv.) was dissolved in 48 mL of ligation buffer in a 50 mL centrifuge tube. The SEQ ID NO:3 solution was added to the thioester solution. The centrifuge tube was rinsed with 2 × 8 mL of ligation buffer (approximately pH 7.0) and added to the reaction mixture. The pH of the reaction mixture was adjusted to approximately 7 with 5 N 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 readjusted to 7.0 with 0.2 mL of 5 N sodium hydroxide solution. The reaction was stirred at room temperature for 24 h and then stored in a freezer. An additional 3 mL of 0.5 M TCEP solution was added before purification. Purification of SEQ ID NO: 5 was performed by RP-HPLC at ambient temperature on a Kromasil C18 10 μm column (10 mm × 250 mm) using a linear gradient of 10% acetonitrile in water for the first 4 min and 20 to 50% acetonitrile in water over 23 min (titrated to 0.1% acetic acid and pH 9.0) during the 28 min purification period. Approximately 372 mg (44.3%) of the tirzepatide cysteine ​​analog SEQ ID NO: 5 was obtained after purification [expected value (mass + 3H]).+ ) / 3=1615.17263, observed value (mass + 3H + ) / 3=1615.1686].

[0139] Desulfurization:

[0140] [ka]

[0141] An aqueous solution of 6-guanidine hydrochloride and 0.3 M disodium hydrogen phosphate monobasic (pH 7.0) was used as the buffer for desulfurization. All solutions were made 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 approximately 7.0 with 5 N NaOH. The volume was brought to 15 mL with buffer. Tirzepatide cysteine ​​analog SEQ ID NO: 5 (105.2 mg, 0.022 mmol) was dissolved in 30 mL of buffer, and 6 mL of Preparation 2 solution was added. 5 mL of 0.3 M glutathione reducing solution (L-GSH, pH 7.0) and 7.5 mL of 0.5 M TCEP solution (pH 7.0) were added. The solution was heated at 44 °C for 4.5 h, after which UPLC analysis showed the reaction was complete [expected (mass + 3H + ) / 3=1604.5153, observed value (mass + 3H + ) / 3=1604.5122]. The desulfurization yield was calculated by UPLC using tirzepatide (SEQ ID NO: 1) reference standard. The yield was estimated to be 47%.

[0142] Example 7 Synthesis of hydrazide fragment 1-20: SEQ ID NO:6

[0143] [ka]

[0144] Hydrazine-CTC resin (2.03 g, 1.32 mmol, loading: 0.65 mmol / g) was placed in a 40 mL reaction vessel and swelled with 3 × 10 mL DCM (30 s each) followed by 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 (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) solution (3.91 mmol, 3.0 equiv.). DIPEA (1.4 mL, 8.04 mmol, 6.1 equiv.) was added to the above solution, and the volume was adjusted to 19 mL with DMF. The solution was mixed on a rotary mixer at room temperature for 30 minutes. 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 x 10 mL of DMF, 5 x 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 in the synthesis of peptide hydrazide SEQ ID NO:6.

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

[0146] Coupling: 3 equivalents of amino acid, 3 equivalents of OXYMA and 3.3 equivalents of DIC were used for amino acid coupling.

[0147] After each coupling and final deprotection cycle, the resin was washed with 5 x 9 mL of DMF for 1 min with N mixing. After peptide hydrazide synthesis, the resin was washed with 7 x 10 mL of DCM for 1 min with N mixing. The resin was then allowed to dry on the synthesizer for approximately 12 h.

[0148] Deprotection and cleavage: 25 mL of cleavage cocktail made with 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 × 2.5 mL), and the filtrate was poured into 175 mL of cold MTBE. The filter flask was washed with TFA (2 × 2.5 mL), and the washes were poured into cold MTBE. After cooling to -20 °C for 30 minutes, the mixture was 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 XSelectCSHC18 10 μm column (10 mm × 250 mm) at ambient temperature. After an initial 3 min of 10% acetonitrile in water, a linear gradient of 20 to 55% acetonitrile in water over 23 min, with 0.1% TFA constant throughout the 28 min purification, was used. Approximately 110 mg of partially purified hydrazide, SEQ ID NO:6, was obtained.

[0149] Deprotection and cleavage: 25 mL of cleavage cocktail made with 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 × 2.5 mL of TFA. The filtrate was poured into 200 mL of cold MTBE, and the peptide was immediately precipitated. The filter flask was then washed with 2 × 2 mL of TFA, and the washings were poured into cold MTBE. After cooling to -20 °C for 30 minutes, the mixture was centrifuged. The peptide precipitate was then washed twice with 240 mL of MTBE and centrifuged. The peptide precipitate was then dried in a vacuum oven at 27 °C for 16 hours. Approximately 1.7 g of crude 19-mer SEQ ID NO:8 was obtained.

[0150] Example 8 Native chemical ligation of fragment 1-20 (SEQ ID NO: 7) to cysteine ​​fragment 21-39 (SEQ ID NO: 8) to form the tirzepatide cysteine-21 analog (SEQ ID NO: 9)

[0151] [ka]

[0152] An aqueous solution of 6 M guanidine hydrochloride and 0.3 M disodium hydrogen phosphate monobasic (pH 7.0) was the ligation buffer used in native chemical ligation. All solutions were made in this ligation buffer. Partially purified peptide hydrazide (SEQ ID NO: 6, 56 mg, 0.019 mmol) was dissolved in 5 mL of ligation buffer (6 M guanidine hydrochloride and 0.3 M disodium hydrogen phosphate monobasic, pH 3.35) and cooled to -15 °C in an acetone-ice bath. 0.25 mL of 1 M sodium nitrite solution (0.25 mmol, 13.2 equiv.) was added to the peptide hydrazide solution and stirred at -15 °C for 10 min. After 10 min, 0.8 mL of 0.5 M 4-mercaptophenylacetic acid (MPAA) solution was added to the peptide hydrazide solution to induce in situ thiolytic cleavage of the peptidyl azide generated from SEQ ID NO: 6. The pH of the reaction mixture was adjusted to about 7.0 with 5N sodium hydroxide solution. Thiol cleavage of the peptidyl azide was carried out for 30 minutes.

[0153] Approximately 0.62 mmol of cysteine ​​peptide 21-39 (SEQ ID NO: 8) was synthesized on Sieber amide resin using a standard SPPS protocol. The N-terminal cysteine-containing SEQ ID NO: 8 (26.1 mg, 0.014 mmol, 0.74 equiv.) 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 minutes, 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 reaction was stirred at room temperature for 1 hour. The tirzepatide cysteine ​​analog (SEQ ID NO: 9) was observed in the reaction mixture.

[0154] Example 9 Native chemical ligation: protected fragment 1-21 (SEQ ID NO: 10) and protected fragment 22-39 (SEQ ID NO: 11)

[0155] [ka]

[0156] Preparation of Reagents / Substrates: 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 μL). 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 μL). A 10% (v / v) solution of DIEA (0.084 mmol, 4.2 eq, 28.64 μL) was prepared in DMF (257.76 μL). A 10 wt% solution of HATU (0.04 mmol, 2.0 eq, 30.418 mg) was prepared in acetonitrile (273.762 μL).

[0157] Coupling and isolation: 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 to it at 0°C, followed by HATU solution. The reaction mixture was stirred at 0°C for 2 hours. 2.0 mL of 17% NaCl / 0.5% was added to it 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 under N2 atmosphere at 40°C.

[0158] Global deprotection: The crude API isolated above was charged with 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). The mixture was stirred at room temperature for 2 hours. It was poured into cold MTBE (30 mL) and kept at 2-4 °C for 30 minutes. It was then centrifuged at 3000 rpm for 3 minutes. The solid precipitate was washed with 2 × 30 mL of MTBE, centrifuging each time. It was dried under vacuum at 40 °C with a N2 purge. 159.13 mg of a white to off-white powder was obtained.

[0159] Example 10 Native chemical ligation: protected fragment 1-17 (SEQ ID NO: 13) and protected fragment 18-39 (SEQ ID NO: 14):

[0160] [ka]

[0161] Preparation of Reagents / Substrates: 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.

[0162] Coupling and isolation: Protected fragment 18-39 (SEQ ID NO: 14) and protected fragment 1-17 (SEQ ID NO: 13) were mixed together at 0°C. DIEA solution and HATU solution were added to the mixture at 0°C. The reaction was stirred at 0°C for 2 hours. To it, 2.0 mL of 17% NaCl / 0.5% was added at 0°C, followed by (2.2 mL) 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 under N2 atmosphere at 40°C.

[0163] Global cleavage: The crude API isolated above was charged with 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). The mixture was stirred at room temperature for 2 hours. It was poured into cold MTBE (30 mL) and kept at 2-4 °C for 30 minutes. It was then centrifuged at 3000 rpm for 3 minutes. The solid precipitate was washed with 2 × 30 mL of MTBE, centrifuged each time, and dried under vacuum at 40 °C with a N2 purge. 159.13 mg of fully deprotected TZP (SEQ ID NO: 1) was obtained as an off-white powder.

[0164] Example 11 Native chemical ligation: Fragment 1-15 (SEQ ID NO: 15) and Fragment 16-39 (SEQ ID NO: 16):

[0165] [ka]

[0166] Preparation of Reagents / Substrates: 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 μL). 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 μL). A 10% (v / v) solution of DIEA (0.084 mmol, 4.2 eq, 14.67 μL) was prepared in DMF (132 μL). A 10 wt% solution of HATU (0.042 mmol, 2.1 eq, 15.209 mg) was prepared in acetonitrile (143.64 μL).

[0167] Coupling and isolation: 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 to it at 0°C, followed by HATU solution. The reaction mixture was stirred at 0°C for 2 hours. 2.0 mL of 17% NaCl / 0.5% was added to it 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 under N2 atmosphere at 40°C.

[0168] Global cleavage: The crude API isolated above was charged with 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). The mixture was stirred at room temperature for 2 hours. It was poured into cold MTBE (30 mL) and kept at 2-4 °C for 30 minutes. It was then centrifuged at 3000 rpm for 3 minutes. The solid precipitate was washed with 2 × 30 mL of MTBE, centrifuged each time, and dried under vacuum at 40 °C with a N2 purge. 159.13 mg of a white to off-white powder was obtained.

[0169] Example 12: Amidation

[0170] [ka]

[0171] Fmoc-GPS(tBu)S(tBu)GAPPPS(tBu)-OH (SEQ ID NO: 18) (1.0625 g, 1 eq.) 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 μL, 1 eq.) was added to the solution. An additional 1 mL of 2-MeTHF was added. Isobutyl chloroformate (0.112 mL, 1 eq.) was added to the reaction mixture, and the mixture was stirred for 10 minutes, after which ammonium hydroxide (0.14 mL, 4 eq.) was added. The resulting mixture was allowed to warm to room temperature and mixed overnight (approximately 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. Actual mass: 1242.6859[M+H].

[0172] Example 13: Fmoc-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-OH (SEQ ID NO: 19)

[0173] [ka]

[0174] Crystallization screening was performed on Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) free form 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 temperature holds, solvent / anti-solvent addition, or a combination of techniques, and non-solvent-based techniques such as thermal stress.

[0175] 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 was a solvated form produced from 1-propanol (1-PrOH) or mixtures containing 1-PrOH (such as 1-PrOH / heptane). Form D was a solvated form produced from acetonitrile (ACN) or mixtures containing ACN (such as ACN / MTBE). Form C was the transformation product of Form D when it was isolated and dried.

[0176] Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form A Preparation 1: 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 amorphous Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) solid, and the sample was stirred at ambient conditions, resulting in a reddish-orange solution. The solution was seeded with 2.8 mg of Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form A and then left at ambient conditions with continued stirring for 2 days. A bright orange suspension was obtained, and the solid was isolated by vacuum filtration using a 10 μm disposable filter, rinsed on the filter twice with 0.5 mL of fresh 1-PrOH, then collected and dried under vacuum at 30 °C for approximately 3-4 hours. The resulting white solid (3.2 grams) was consistent with Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form A.

[0177] Preparation 2: 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 heptane to obtain a clear, pale yellow solution. With stirring, an additional 0.6 mL of heptane was added to the solution, and the sample was capped and stirred at ambient conditions for 2 days. A suspension was obtained, and the solid was consistent with Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form A.

[0178] Preparation 3: 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 at ambient conditions for 1 day and then transferred to a freezer for 3 days. The solid observed in the solution was consistent with Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form A.

[0179] XRPD of Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form A The XRPD pattern of crystalline Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form A was obtained on a Bruker D8 Endeavor X-ray powder diffractometer equipped with a CuKα (1.5418 Å) source and a Linxeye detector, operating at 40 kV and 40 mA. The sample was scanned from 4 to 42° 2θ, with a step size of 0.009° 2θ and a scan rate of 0.5 seconds per step, using a 0.3° primary slit aperture and a 3.9° particle size distribution (PSD) aperture. The powder was packed into a quartz sample holder, and a smooth surface was obtained using a glass slide. Diffraction patterns were collected at ambient temperature and relative humidity. Crystalline peak positions were determined using MDI-Jade v7.9.9.

[0180] A prepared sample of Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form A is characterized by an XRPD pattern using CuKα radiation as having diffraction peaks (2-theta values) as set forth in Table 1 below (including a peak at 6.1° 2-theta in combination with one or more peaks selected from 8.5, 11.7, 12.3, and 16.9° 2-theta), with a diffraction angle tolerance of 0.2 degrees. A representative XRPD pattern of Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form A is shown in Figure 1A.

[0181] [Table 1]

[0182] Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form C Preparation 1: 0.5 mL of ACN was added to 66.9 milligrams of Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form A, and the sample was stirred at 51 °C, resulting in a clear, colorless solution. The solution was transferred 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 at ambient conditions for 3 days. A white suspension was obtained, and the solid was isolated using a 0.45 μm nylon syringe filter and centrifuged at ambient conditions for 5 minutes. The white solid on the filter was allowed to stand at ambient conditions and air-dried under a gentle stream of N2 for approximately 20 minutes. The resulting white solid is consistent with Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form C.

[0183] Preparation 2: Eight milliliters of 2:1 v / v 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, resulting in a white suspension. The suspension was seeded with Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form C and then left at ambient conditions with continued stirring for 2 days. The solid was isolated by vacuum filtration using a 10 μm disposable filter, rinsed on the filter with 1 mL of fresh 2:1 v / v ACN / MTBE, collected, and dried under vacuum at 30° C. The dried white solid was consistent with Fmoc-GPS(tBu)-S(tBu)-G-OH Form C.

[0184] XRPD of Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form C 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.

[0185] A prepared sample of Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form C is characterized by an XRPD pattern using CuKα radiation as having diffraction peaks (2-theta values) as set forth in Table 2 below (including a peak at 6.7° 2-theta in combination with one or more peaks selected from the group consisting of 8.7, 10.6, 14.1, and 15.8° 2-theta), with a diffraction angle tolerance of 0.2 degrees. A representative XRPD pattern of Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form C is shown in Figure 1B.

[0186] [Table 2]

[0187] Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form D Preparation 1: One mL of 1:1 v / v ACN / MTBE was added to 72.6 milligrams of Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form A, and the sample was stirred at ambient conditions, resulting in a white suspension. The suspension was seeded with Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form C and then left at ambient conditions with continued stirring for 2 days. The wet solid from the slurry was consistent with Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form D, which was not physically stable and converted to Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form C upon isolation and drying.

[0188] XRPD of Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form D 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 degrees at a scan rate of 0.1 sec / step.

[0189] A prepared sample of Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form D is characterized by an XRPD pattern using CuKα radiation as having diffraction peaks (2-theta values) as set forth in Table 3 below (including a peak at 6.1° 2-theta in combination with one or more peaks selected from the group consisting of 4.1, 10.4, and 12.7° 2-theta), with a diffraction angle tolerance of 0.2 degrees. A representative XRPD pattern of Fmoc-GPS(tBu)-S(tBu)-G-OH (SEQ ID NO: 19) Form D is shown in Figure 1C.

[0190] [Table 3]

[0191] Example 14: Gelation studies The gelation properties of the peptide fragments were investigated under a variety of conditions, including different process solvents (DMSO / ACN and DMF), peptide fragment concentrations, gelation over time, process shear rate (by varying the cross-flow rate), temperature conditions, and gelation reversibility.

[0192] Process Solvents The gelation properties of the peptide fragments were investigated in DMSO / ACN and DMF at various peptide fragment concentrations. Viscosity measurements (Pa.s) at a shear rate of 1 / s are shown in Figure 2A. The results showed that the peptide products in DMF became more viscous at lower concentration values ​​when compared to DMSO / ACN.

[0193] Concentration and time The gelation properties of the peptide fragments were investigated in DMSO / ACN and DMF at various peptide fragment concentrations over a 4-day period. Viscosity measurements (Pa.s) at a shear rate of 1 / s are shown in Figure 2B. The results showed that the viscosity of the formulations increased with peptide fragment concentration and with time in both solvent systems.

[0194] shear rate The gelation properties of the peptide fragments were investigated at various shear rate conditions for peptide fragment concentrations of 40 mg / mL, 50 mg / mL, 60 mg / mL, and 70 mg / mL in DMF. Viscosity measurements (Pa.s) are shown in Figure 2C. The results showed that the peptide fragments at 40 mg / mL and 50 mg / mL concentrations in DMF maintained consistent viscosities with increasing shear rate, while the peptide fragments at 60 mg / mL and 70 mg / mL concentrations in DMF exhibited a significant decrease in viscosity with increasing shear rate.

[0195] The gelation properties of the peptide fragments were investigated at various shear rate conditions for peptide fragment concentrations of 50 mg / mL, 60 mg / mL, and 70 mg / mL in DMSO / ACN. Viscosity measurements (Pa.s) are shown in Figure 2D. The results showed that the peptide fragments at 50 mg / mL and 60 mg / mL concentrations in DMSO / ACN maintained consistent viscosities with increasing shear rate, while the peptide fragment at 70 mg / mL concentration in DMSO / ACN exhibited a significant decrease in viscosity with increasing shear rate.

[0196] Parameter Range Using the results from the gelation studies, improved parameter conditions (temperature, concentration, shear rate, cross-flow velocity) for nanofiltration of the peptide fragments of the present disclosure were investigated and defined. The improved parameter conditions included: (i) a temperature of 10 to 34°C (target of approximately 20°C), (ii) a turbulence / cross-flow velocity of 1.94 to 2.46 m / s (target of approximately 2.2 m / s), (iii) a viscosity of 2.1 x 10 3~2.7×10 3 Laminar flow / shear rate of 1 / s (target approximately 2.4 x 10 3 1 / s), (iv) a primary concentration target of 32.8 to 47.2 mg / mL (target approximately 40 mg / mL), and (v) a secondary concentration target of 37 to 53 mg / mL (target approximately 45 mg / mL).

[0197] High temperatures, low peptide fragment concentrations, high shear rates and high cross-flow velocities can be used to reduce viscosity and therefore reverse gelation.

[0198] array SEQ ID NO: 1 - Tirzepatide

[0199] [ka] where K at position 20 is the epsilon-amino group of the K side chain and (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γGlu)1-CO—(CH2) 18 It is chemically modified by conjugation with -CO2H.

[0200] SEQ ID NO:2 - Hydrazide fragment 1-17

[0201] [ka]

[0202] SEQ ID NO:3-Cysteine-18 fragment 18-39

[0203] [ka]

[0204] SEQ ID NO:4 - Thioester fragment 1-17

[0205] [ka]

[0206] SEQ ID NO:5 - TZP cysteine-18 analog

[0207] [ka]

[0208] SEQ ID NO:6 - Hydrazide fragment 1-20

[0209] [ka]

[0210] SEQ ID NO:7 - Thioester fragment 1-20

[0211] [ka]

[0212] SEQ ID NO:8-Cysteine-21 fragment 21-39

[0213] [ka]

[0214] SEQ ID NO:9 - TZP cysteine-21 analog

[0215] [ka]

[0216] SEQ ID NO: 10 - Protected fragment 1-21

[0217] [ka]

[0218] SEQ ID NO: 11 - Protected fragment 22-39

[0219] [ka]

[0220] SEQ ID NO: 12 - Protected TZP

[0221] [ka]

[0222] SEQ ID NO: 13 - Protected fragments 1-17

[0223] [ka]

[0224] SEQ ID NO: 14 - Protected fragment 18-39

[0225] [ka]

[0226] SEQ ID NO: 15 - Protected fragment 1-15

[0227] [ka]

[0228] SEQ ID NO: 16 - Protected fragment 16-39

[0229] [ka]

Claims

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

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

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

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

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

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

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

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

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

13. A compound of SEQ ID NO: 10, or a pharmaceutically acceptable salt thereof, comprising one or more protecting groups.

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

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

10.

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

10.

17. A compound of SEQ ID NO: 13, or a pharmaceutically acceptable salt thereof, comprising one or more protecting groups.

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

19. A compound of SEQ ID NO: 14, or a pharmaceutically acceptable salt thereof, comprising one or more protecting groups.

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

21. A compound of SEQ ID NO: 15, or a pharmaceutically acceptable salt thereof, comprising one or more protecting groups.

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

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

15.

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

15.

25. A compound of SEQ ID NO: 16, or a pharmaceutically acceptable salt thereof, comprising one or more protecting groups.

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

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

16.

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

16.

29. A process for preparing a compound of formula (I), comprising: 【Chemistry 1】 wherein PG is a protecting group and the process comprises: (a) contacting a compound of formula (Ia) with diisopropylethylamine; 【Chemistry 2】 (b) contacting the product of step (a) with 2-(5-norbornene-2,3-dicarboximido)-1,1,3,3-tetramethyluronium tetrafluoroborate in DMF; (c) adding a compound of formula (Ib) to the mixture of (b); 【Transformation 3】 The process includes:

30. 30. The process of claim 29, wherein the protecting group is selected from Boc and Fmoc.

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

32. The process of any one of claims 29 to 31, wherein the process is carried out using continuous flow.

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

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