Method for preparing GIP / GLP1 dual agonists

Abstract

The invention relates to a method for preparing a GIP / GLP1 dual agonist. In particular, the present invention provides novel intermediates and processes useful in the manufacture of tizatide or a pharmaceutically acceptable salt thereof.

Description

This application is a divisional application of the invention patent application with the application date of January 28, 2020, application number 202080011536.7, and name “Method for Preparing GIP / GLP Dual Agonist”.

[0001] The present invention provides a method and intermediates for preparing the GIP / GLP1 dual agonist peptide tirzepatide or a pharmaceutically acceptable salt thereof.

[0002] Diabetes is a chronic disease characterized by high blood sugar levels caused by 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 blood sugar levels. U.S. Patent 9,474,780 (the "'780 patent") describes and claims the dual GIP / GLP1 agonist tirzepatide. Tirzepatide can be used to treat T2D.

[0003] US9474780 generally describes peptides and methods of making GIP / GLP1 dual agonists.

[0004] There is a need for methods and intermediates to improve the technology for producing tirzepatide, so that the technology has multiple advantages, including commercially desirable purity. Similarly, there is a need for efficient and environmentally friendly "green" methods, including stable intermediates, to provide tirzepatide with fewer purification steps. To improve environmental and operator safety, there is also a need for improved technologies to provide tirzepatide preparation methods that generate minimal waste streams. The preparation of large-scale, pharmaceutically elegant tirzepatide presents many technical challenges that may affect overall yield and purity. There is a need for methods that avoid the use of transition metals and / or harsh reaction conditions that are incompatible with peptide synthesis.

[0005] The present invention seeks to address these needs by providing novel intermediates and methods useful for preparing tirzepatide (SEQ ID NO: 1) or a pharmaceutically acceptable salt thereof. The improved methods for preparing tirzepatide of the present invention provide intermediates and process reactions that embody a combination of advantages, including an efficient route with fewer steps, while maintaining high quality and purity. Importantly, the improved methods and intermediates reduce resource intensity and minimize waste streams.

[0006] The improved processes described herein provide various embodiments of intermediates that are useful in the production of tirzepatide.

[0007] The present invention provides a compound of SEQ ID NO: 17 or a pharmaceutically acceptable salt thereof. The present invention provides a compound of SEQ ID NO: 11 or a pharmaceutically acceptable salt thereof. The present invention provides a compound of SEQ ID NO: 22 or a pharmaceutically acceptable salt thereof. The present invention provides a compound of SEQ ID NO: 21 or a pharmaceutically acceptable salt thereof. The present invention provides a compound of SEQ ID NO: 20 or a pharmaceutically acceptable salt thereof. The present invention provides a compound of SEQ ID NO: 2 or a pharmaceutically acceptable salt thereof. The present invention provides a compound of SEQ ID NO: 4 or a pharmaceutically acceptable salt thereof. The present invention provides a compound of SEQ ID NO: 7 or a pharmaceutically acceptable salt thereof. The present invention provides a compound of SEQ ID NO: 14 or a pharmaceutically acceptable salt thereof. The present invention provides a compound of SEQ ID NO: 33 or a pharmaceutically acceptable salt thereof. The present invention provides a compound of SEQ ID NO: 32 or a pharmaceutically acceptable salt thereof. The present invention provides a compound of SEQ ID NO: 34 or a pharmaceutically acceptable salt thereof. The present invention provides a compound of SEQ ID NO: 35 or a pharmaceutically acceptable salt thereof. The present invention provides a compound of SEQ ID NO: 36 or a pharmaceutically acceptable salt thereof. The present invention provides a compound of SEQ ID NO: 38 or a pharmaceutically acceptable salt thereof. The present invention provides a compound of SEQ ID NO: 39 or a pharmaceutically acceptable salt thereof.

[0008] The present invention provides compounds of the following formula: or a pharmaceutically acceptable salt thereof.

[0009] The present invention provides compounds of the following formula: or a pharmaceutically acceptable salt thereof.

[0010] The present invention provides a process wherein tirzepatide is prepared using nanofiltration.

[0011] The present invention provides a method for preparing tirzepatide, comprising deprotecting the compound of SEQ ID NO: 22 or a pharmaceutically acceptable salt thereof.

[0012] The present invention provides a method for selectively acylating a lysine amino acid, wherein the lysine amino acid and the N-terminus are protected. The present invention provides a method for selectively acylating a lysine amino acid in a peptide, comprising coupling a resin-bound peptide-lysine-NH2 with tert-butyl-eicosanedioyl-Glu-(O-tert-butyl)-(8-amino-3,6-dioxaoctanoic acid)-(8-amino-3,6-dioxaoctanoic acid)-OH. The present invention provides a method for preparing tirzepatide, comprising deprotecting a compound of SEQ ID NO: 22 or a pharmaceutically acceptable salt thereof.

[0013] The present invention provides a method for deprotecting tirzepatide, wherein the deprotection solution comprises dithiothreitol, triisopropylsilane and trifluoroacetic acid.

[0014] The present invention provides a method for selectively acylating lysine amino acids, wherein the resin-bound peptide-lysine-NH2 is a compound of the formula: or a pharmaceutically acceptable salt thereof.

[0015] The present invention provides a method for converting a depsipeptide isomer into a desired peptide, comprising: adjusting the pH of the depsipeptide isomer to about pH 7 to about pH 10; and incubating the depsipeptide isomer at pH 7 to pH 10 for at least one hour.

[0016] The present invention provides a method for converting a depsipeptide isomer, wherein the depsipeptide isomer is adjusted to about pH 8.5 to about pH 9.5.

[0017] The present invention provides a method for converting a depsipeptide isomer, wherein the depsipeptide isomer is a compound represented by SEQ ID NO: 40 or a pharmaceutically acceptable salt thereof.

[0018] The present invention provides a free radical-based desulfurization comprising contacting a peptide with a free radical initiator. In one embodiment, the desulfurization comprises contacting a peptide suitable for desulfurization with a water-soluble free radical initiator. In one embodiment, the free radical initiator is an azo initiator. In one embodiment, the free radical initiator is selected from 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (VA-044) and 2,2'-azobis(2-methylpropionamidine) dihydrochloride (VA-050).

[0019] The free radical-based desulfurization methods provided herein are environmentally desirable, transition metal-free, and compatible with peptide synthesis conditions.

[0020] As used herein, the following abbreviations have the meanings as given herein: “SPPS” means solid phase peptide synthesis, “Fmoc” means fluorenylmethoxycarbonyl chloride, “Pip” means piperidine, “DIC” means diisopropylcarbodiimide, “Oxyma” means ethyl cyanohydroxyimidoacetate, “DCM” means dichloromethane, “IPA” means isopropyl alcohol, “MTBE” means methyl tert-butyl ether, “TFA” means trifluoroacetic acid, “TIPS” means triisopropylsilane, “DTT” means dithiothreitol, “UPLC” means ultra performance liquid chromatography, and “HFIP” means hexafluoroisopropyl ether. alcohol, "CTC" means trityl chloride, "HATU" means (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, "TFET" means 2,2,2-trifluoroethanethiol, "DIEA" means N,N-diisopropylethylamine, "AEEA" means 17-amino-10-oxo-3,6,12,15-tetraoxa-9-aza-heptadecanoic acid, "TCEP" means tris(2-carboxyethyl)phosphine, "DCU" means dicyclohexylurea, "DCC" means dicyclohexylcarbodiimide, "TM SA" means trimethylsilyalmide, "HOBt" means hydroxybenzotriazole, "HRMS" means high-resolution mass spectrometry, "LPPS" means liquid phase peptide synthesis, "MSMPR" means mixed product mixed suspension reactor, "MPA" means mobile phase A, "MPB" means mobile phase B, "L-GSH" means L-glutathione reduced solution, "TZP" means tirzepatide, "AP" means active pharmaceutical ingredient, and "API" means active pharmaceutical ingredient, "PyBOP" means (benzotriazole-1- "DEA" means diethylamine, "TBTU" means 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylammonium tetrafluoroborate, "TNTU" means 2-(5-norbornene-2,3-diimido)-1,1,3,3-tetramethyluronium tetrafluoroborate, "PyOxim" means 1-cyano-2-ethoxy-2-oxoethylideneaminooxy-tripyrrolidinophosphonium hexafluorophosphate, and "PyClock" means 6-chloro-benzotriazol-1-yloxy-tripyrrolidinophosphonium hexafluorophosphate. As shown herein, amino acid one-letter abbreviations are shown in bold, while atoms are shown in non-bold text and generally in a smaller font to distinguish them from the one-letter amino acid abbreviations. As used herein, when an amino acid abbreviation appears with a number above the amino acid, the number refers to the corresponding amino acid position in the final tirzepatide 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 indicated in such sequence.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 method.

[0021] Those skilled in the art will appreciate that there are alternative resins for constructing the peptides shown herein. For example, Sieber and Rink amide resins are well known to those skilled in the art for preparing the peptides disclosed herein; however, alternative resins may be selected for use in preparing the peptides described herein. For example, but not limited to, 2-CTC and related resins may be used to prepare the target peptide, followed by a C-terminal amidation step.

[0022] Solid-phase peptide synthesis (SPPS) construction was accomplished using standard fluorenylmethyloxycarbonyl chloride (Fmoc) peptide chemistry techniques employing sequential couplings with an automated peptide synthesizer. The resin was swollen with DMF and then deprotected using 20% ​​piperidine (Pip) / DMF (3 x 30 min). Subsequent Fmoc deprotection used 20% Pip / DMF for 3 x 30 min, with 4 x 30 min treatments for more difficult couplings. Following deprotection, the resin was washed with 5 x 2 min, 10 volume washes of DMF. Amino acid preactivation was performed using diisopropylcarbodiimide (DIC) / ethyl cyanohydroxyiminoacetate (Oxyma) in DMF at room temperature for 30 min. Coupling of the activated amino acid to the resin-bound peptide occurred at a specific time for each individual amino acid. After each coupling, solvent washes were performed using 5 x 2 min, 10 volume washes of DMF. To isolate the final product, the resin-bound product was washed with 10 volumes of DCM for 5 x 2 min to remove the DMF. The resin was washed with 2 × 2 min 10 volumes of IPA to remove DCM, washed with 5 × 2 min 10 volumes of methyl tert-butyl ether (MTBE), and then the product was dried under vacuum at 40 ° C. The resin-bound product was refrigerated (-20 ° C). For analysis, the peptide was cleaved from the resin using an acidic mixture consisting of trifluoroacetic acid (TFA) / H2O / TIPS (triisopropylsilane) / DTT (dithiothreitol) in the following ratios: (0.93v / 0.04v / 0.03v / 0.03w). The resin was swollen with DCM (4-5mL, 3 × 30min) and drained. The cleavage mixture (4-5mL) was added to the pre-swollen resin, and the suspension was stirred at room temperature for 2 hours. The solution was filtered, then the resin was washed with a small amount of DCM and combined with the cleavage solution. The resulting solution was poured into 7-10 volumes of cold (0 ° C) methyl tert-butyl ether (MTBE). The suspension was aged at 0°C for 30 minutes, after which the resulting precipitate was centrifuged and the clear solution was decanted. The residue was suspended in the same volume of MTBE, and the resulting suspension was centrifuged again and decanted. After decanting, the clear MTBE solution of the precipitated peptide was dried overnight under vacuum at 40°C.

[0023] Synthesis of Preparation 1: SEQ ID NO: 2 The synthesis used Fmoc-Sieber amide resin with a loading of 0.71 mmol / g. The general SPPS protocol was used with the following modifications:

[0024] Preparation 1 Soft Cleavage: Ten identical deprotection reactions were performed in parallel, each with a ~0.5 mmol scale of resin-bound Preparation 1, using the following protocol: 1) Add 1.55 g (~0.5 mmol) of resin-bound Preparation 1 to a 40 mL fritted reactor. 2) Swell with 3 x 15 mL of DMF (15 min each), 3) Treat with 3 x 15 mL (30 min each) of 20% Pip / DMF. 4) Wash with 4 x 15 mL of DMF, followed by 4 x 15 mL of DCM. 5) Add 1.5 mL of TFA and 28.5 mL of DCM to each of five 40 mL reaction vials. 6) Add one-fifth of Preparation 1 resin-bound (2.75 g) to each of the TFA solution vials, cap the vials, and mix on a rotating wheel for 5 minutes. 7) Filter the mixture and wash with 100 mL of DCM to give a total filtrate volume of 500 mL. 8) The filtrates were combined and transferred to a round-bottom flask containing 1000 mL of MTBE. 9) The resulting suspension was concentrated to a light yellow oil, triturated with 200 mL of MTBE, and cooled in an ice bath for 30 minutes. 10) The solid was filtered, washed with 50 mL of cold MTBE, and dried in a vacuum oven at 33° C. overnight to produce 5.35 g (91% yield) of a white solid. The isolated solid was analyzed using UPLC (98.57 area %, with 0.99% of the combined t-Bu deprotection byproduct).

[0025] Synthesis of Preparation 2: SEQ ID NO: 3 The synthesis used Fmoc-Gly-OH 2-CTC resin with a loading of 0.61 mmol / g. The general SPPS protocol was used with the following modifications:

[0026] Preparation 2 soft cleavage: to a 40 mL glass scintillation vial, resin-bound Preparation 2 (3.06 g, 1.12 mmol) and 30 mL of 30% HFIP DCM solution were added, wherein a red color change was observed. The vial was agitated by rotating on a wheel at room temperature for 1 hour. The resin was filtered off and washed with 3 × 10 mL DCM. The solvent was removed in vacuo to form a glassy foam (35 ° C bath, 10 torr, 2.34 g), and replaced with a small proportion of IPA (24 mL), and then water (24 mL) was added dropwise at room temperature over 25 min. The resulting solution was stirred for 30 min and then filtered. The filter cake was washed with 3 × 10 mL H2O and then dried overnight in a vacuum oven at 25 torr and 35 ° C. This produced Preparation 2 as a white solid (1.81 g).

[0027] Synthesis of Preparation 4: SEQ ID NO: 4 The synthesis used Fmoc-Leu-OH 2-CTC resin with a loading of 0.68 mmol / g. The general SPPS protocol was used with the following modifications:

[0028] Preparation 4 soft cleavage: To a 20 mL glass scintillation vial was added resin-bound Preparation 4 (2.0 g, 0.62 mmol) and 10 mL of a 30% HFIP DCM solution, where a red color change was observed. The vial was agitated by rotating on a wheel at ambient temperature, then the resin was filtered out, washed with 3 x 2 mL DCM, and the solvent was removed in vacuo to form a glassy, ​​viscous foam. The foam was dissolved in 5.2 mL DMSO. The solution with 6 mL of water was added with 1 mL of water at an equal flow rate (T~15°C) over 45 minutes. Once the peptide solution was completely added, an additional 6 mL of water was added over 45 minutes. A white solid precipitated after the addition. The resulting slurry was stirred at 15°C for 30 minutes. The solid was filtered, washed with 6 mL of water, and then transferred to a vacuum oven at 35°C and 25 torr. This produced Preparation 4 (Boc-1-14-OH, 1.0763 g) as a white, fluffy solid.

[0029] Synthesis of Preparation 3 using LPPS: SEQ ID NO: 5 Preparation 2 (500 mg, 0.183 mmol), Preparation 1 (179 mg, 0.175 mmol) and DMSO (10 mL) were added to a 20 mL glass scintillation vial. DIEA (46 μL, 0.265 mmol) was added to the solution, followed by PyBOP (benzotriazole-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate) (123 mg, 0.230 mmol). The reaction was then stirred for 2 hours, diethylamine (DEA) (183 microliters, 1.77 mmol) was added, and the resulting solution was stirred for 2 hours. The contents of the reaction were drawn out into a syringe, added to a stirred 50 mL flask over 1 hour, and water (12 mL) was added dropwise. After the addition was complete, the precipitated product was collected by filtration and then washed with water (2 × 4 mL). The wet cake was dried at 35 ° C under vacuum for 18 hours to obtain Preparation 3 as a white solid (0.6003 g, 88% yield, HRMS calculated C 184 H 261 N 31 O 38 The expected value is 3512.9444, and the measured value is 3512.9430).

[0030] Synthesis of Preparation 5 using LPPS: SEQ ID NO: 6 Preparation 3 (338.8 mg, 0.091 mmol), Preparation 4 (192.1 mg, 0.091 mmol) and DMSO (10 mL) were added to a 20 mL glass scintillation vial. PyBOP (63.5 mg, 0.118 mmol) was added to the solution, followed by DIEA (79 microliters, 0.454 mmol). The reaction solution was stirred for 2.5 hours. The contents of the reaction were drawn out into a syringe and added to a stirred 50 mL flask over 1 hour while water (12 mL) was added dropwise. After the addition was complete, the precipitated product was collected by filtration and subsequently washed with water (2 × 4 mL). The wet cake was dried under vacuum at 35 ° C for 18 hours to obtain Preparation 5 as a white solid (0.3568 g, 70% yield, HRMS calculated C 293 H 435 N 45 O 64 The expected value is 5608.2168, and the measured value is 5608.2066).

[0031] Synthesis of Preparation 6 using Method 1 (LPPS) Docosanoic acid mono(1,1-dimethylethyl) ester (15.0 kg, limited reagent) and N-hydroxy-succinimide (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 out, and the organic phase was then extracted 3 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 provide 17.0 kg of INT1, which was 87% yield and 99% purity.

[0032] H-Glu-OtBu (7.7 kg, 1.1 eq) was dissolved in DCM (54 L) at 20 ° C, followed by addition of a solution of TMSA (11.3 kg) dissolved in DCM (7 L), and the reaction mixture was stirred at 40 ° C for 1 hour. INT1 (17.0 kg) DCM solution was added at room temperature and stirred for 8 hours. After completion of the reaction, DCM was exchanged for ethyl acetate by distillation. The organic phase was washed 3 times with a 2% KHSO4 / NaCl aqueous solution and then 4 times with a 2% NaCl aqueous solution. After separation and removal of 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 mixture of water / isopropanol and then dried at 30 ° C to produce 17.3 kg of INT 2, which was 86% yield and 99% purity.

[0033] INT 2 (17.3 kg) and N-hydroxy-succinimide (4.1 kg, 1.2 eq) were dissolved in ethyl acetate (336 kg) at 27 ° C. A solution of DCC (8.33 kg, 1.25 eq) was added and the reaction was stirred at 22 ° C for 24 hours. The DCU by-product was filtered out. The organic phase was concentrated, co-evaporated with isopropanol, and then crystallized by cooling isopropanol solution (~125 L). The filter cake was then rinsed with cold isopropanol and dried at 25 ° C to provide 16.3 kg INT 3, which was 81% yield and 96% purity.

[0034] 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. INT 3 (16 kg) was suspended in DCM (31 L) at 35 ° C and added to the TMS-protected (AEEA2) mixture at 22 ° C. The reaction was stirred for 12 hours, and after the reaction was complete, the mixture was concentrated and then exchanged into ethyl acetate. The organic phase was washed three times with a 2% KHSO4 / NaCl aqueous solution (~200 L), and then washed four times with a 2% NaCl aqueous solution (~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 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 provide 18.4 kg of Preparation 6 (88% yield) with 96% purity. Overall yield = 53%.

[0035] Synthesis of Preparation 6 using Method 2 (SPPS) Alternatively, Preparation 6 can be prepared using solid phase peptide synthesis using a peptide synthesizer.

[0036] Standard coupling procedures were used.

[0037] Standard coupling conditions: 0.133 M, 2.0 eq. HATU, 5.0 eq. DIEA, ambient temperature, 3 hours, deprotected with 20% piperidine / DMF 3 x 15 min.

[0038] Resin feeding: FmocNH-AEEA on 2-CTC resin (0.99 mmol / g): 1.01 g in each parallel reaction.

[0039] The automated program used a DMF swell followed by Pip / DMF; a DMF wash; and an amino acid, DIEA, HATU mixture; and a DMF wash cycle followed by drying.

[0040] The resin was stirred in 30% HFIP / DCM (240 mL) for 1.5 hours and cracked. The resin was filtered, washed, and the solvent was removed from the filtrate in a vacuum. The gained oil was dissolved in acetonitrile and the solvent was removed again. This operation provided 30.47 g (146% of theoretical yield) of viscous yellow oil, which contained 52.3 area % of the desired product analyzed by UPLC. The crude product was purified by flash chromatography (500 grams of silica gel, eluted with 85% DCM / 10% methanol / 5% acetic acid, 38 × 100 mL fractions were collected). The previous chromatographic concentrate (17.94 g) was crystallized to produce 13.4 g (74.7% yield), the UPLC purity of which was 91.65 area %.

[0041] Example 1 Synthesis Example 1 SEQ ID NO: 1 To the first HPLC vial was added Preparation 5 (10.5 mg, 0.00187 mmol) and DCM (200 μL, 20 L / kg). To this solution was added a solution of phenylsilane (0.81 M in DCM, 22.1 μL, 0.0178 mmol) and tetrakis(triphenylphosphine)-palladium(0) (0.8 M in DCM, 22.1 μL, 0.00064 mmol). The solution was stirred at 24° C. for one hour to give a non-separated solution of Preparation 7 (SEQ ID NO: 7). To the second HPLC vial was added DCM (150 μL), followed by Preparation 6 (0.118 M in DCM, 16 μL, 0.00189 mmol), PyBOP (0.186 M in DCM, 16 μL, 0.00298 mmol) and DIEA (0.573 M in DCM, 5 equivalents). The contents of the second vial were added to the first vial and the reaction was stirred for 1 hour to give a non-isolated solution of Preparation 8 (SEQ ID NO: 8). The solution of Preparation 8 was concentrated under vacuum to give a solid, to which 50 μL of a solution of trifluoroacetic acid (4.65 mL), triisopropylsilane (20 μL) and DTT (20 mg) were added. The slurry was stirred for 18 hours and monitored by HPLC to confirm the formation of Example 1 (HRMS calculated C 225 H 348 N 48 O 68 The expected value is 4810.5249, and the measured value is 4810.5257).

[0042] Synthesis of Preparation 9 SEQ ID NO: 9 Sieber amide resin (13.42g, 0.75mmol / g, 10.1mmol) is suspended in DMF (130mL, 10 volumes) about 20min, then drains.With DMF (80mL, 6 volumes) washing gained resin about 5min.By with 5 volume % piperidines, 1.25 volume %DBU, 1.0wt.%HOBt / DMF solution (80mL, 6 volumes) handle Fmoc-amino acid resin twice (being respectively 10min and 20min) remove Fmoc group.After draining off-Fmoc solution, with DMF (80mL, 6 volumes) twice, MTBE (80mL, 6 volumes) twice and again with DMF (80mL, 6 volumes) twice washing.

[0043] Amino acid chains were assembled using standard Fmoc chemistry. Generally, 1.5 equivalents of Fmoc-amino acid and HOBt (2.47 g, 20% water wetness, 14.6 mmol, 1.46 equivalents) were dissolved in DMF (60 mL, 4.5 volumes), followed by the addition of DIEA (1.94 mL, 11.1 mmol, 1.11 equivalents). The resulting solution was cooled to <5°C with an ice bath and activated by the addition of TBTU (4.83 g, 15.0 mmol, 1.5 equivalents). The mixture was allowed to stand at 0°C-5°C for approximately 5 minutes. DCM (60 mL, 1.5 volumes) was added to the resin, followed by the addition of activated Fmoc-amino acid solution. The resulting mixture was stirred at approximately ambient temperature for 2 hours. The Fmoc removal process was repeated and the remaining amino acid was coupled in sequence. After the final Fmoc removal step was completed, the resin was washed twice with 2-propanol (130 mL, 10 volumes) for 5 min, followed by six washes with MTBE (130 mL, 10 volumes). The resin was dried in vacuo at 35°C to afford Preparation 9-Seiber (21.21 g, 0.435 mmol / g theoretical, 91.7% yield based on mass gain).

[0044] A portion of Preparation 9-resin complex (10.15 g, 0.435 mmol / g, 4.41 mmol) was treated with a solution of 5% by volume TFA in DCM (101 mL, 10 volumes) and a DCM wash step. The cleavage fraction and wash were neutralized with DIEA (26.29 g, 35.5 mL, 1.01:1 molar ratio to TFA). The fractions were combined and concentrated under vacuum to 50% of the original volume. The DCM solution was washed with saturated aqueous NaHCO₃ (2×94 mL). The resulting solution was dried over anhydrous MgSO₄ and concentrated to dryness to produce a sticky solid. The sticky solid was reslurried in <5°C MTBE (100 mL) to break up the gum, yielding the product as a white slurry. The white powder slurry was filtered, washed and dried to give Preparation 9 (3.84 g, 92.3 area%, 37.8 wt% DIEA·TFA, 57.4 wt%, 2.29 mmol, 51.9% yield, HRMS calculated C 46 H 78 N 10 O 12 The expected value is 962.5801, the measured value is 962.5806), and it is a white powder.

[0045] Synthesis of Preparation 10 SEQ ID NO: 10 Fmoc-Gly-Gly-O-2CTC resin complex (18.09g, 0.57mmol / g, 10.3mmol) is suspended in DMF (180mL, 10 volumes) middle 20min, drain then.With DMF (108mL, 6 volumes) washing gained resin 5min.By with 5 volume % piperidines, 1.25 volume %DBU, 1.0wt.%HOBt / DMF solution (108mL, 6 volumes) handle Fmoc-amino acid resin twice (being respectively 10min and 20min) remove Fmoc group.Drain and take off-Fmoc solution, and with DMF (110mL, 6 volumes) twice, MTBE (110mL, 6 volumes) twice and use DMF (110mL, 6 volumes) twice washed resin again.Chain assembly is carried out with standard Fmoc chemistry.

[0046] For the coupling of amino acids, generally 1.5 equivalents of Fmoc-amino acid and HOBt (2.54 g, 20% water wet, 15.0 mmol, 1.5 equivalents) were dissolved in DMF (80 mL, 4.4 volumes), followed by the addition of DIEA (1.94 g, 15.0 mmol, 1.5 equivalents) to provide the coupling of amino acids. The resulting solution was cooled to 0-5 ° C with an ice bath and activated by the addition of 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylammonium tetrafluoroborate (TBTU) (4.84 g, 15.1 mmol, 1.5 equivalents). It was allowed to stand at 0-5 ° C for 5 min. DCM (35 g, 1.5 volumes) was then added to the resin, followed by the addition of the activated Fmoc-amino acid solution. The resulting mixture was stirred at room temperature for 2 hours. After completing the synthesis steps, the peptide resin was washed with 2-propanol (180 mL, 10 volumes) for 5 min twice, then washed with MTBE (180 mL, 10 volumes each, 6 times), and then dried at 35°C to obtain Preparation 10 resin complex (25.52 g, 0.216 mmol / g, 53.6% yield).

[0047] A portion of Preparation 10 resin complex (10.075 g, 0.216 mmol / g, 2.18 mmol) was treated three times with 1% by volume of TFA in DCM (100 mL, 10 volumes) and washed with DCM (75 mL, 7.5 volumes). The cleavage fractions and washings were neutralized with pyridine (3.18 g, 1.01:1 molar ratio to TFA). The fractions were combined and concentrated to dryness under vacuum at ≤35°C. Reconstitution was performed with ethanol (40 mL, 10% by volume of the combined filtrate) followed by concentration to dryness. Finally, the peptide was triturated with stirring in deionized water (150 mL, 40% by volume of the combined filtrate). The solid crude peptide precipitate was collected by centrifugation and washed twice with deionized water (150 mL each). The solid was washed twice with n-heptane (100 mL each time), isolated, and dried under vacuum at 40° C. to yield Preparation 10 (SEQ ID NO: 10) as a brittle, light yellow solid (4.10 g, 72.4 area%, 3.0 wt% pyridine·TFA, 70.2 wt%, 1.85 mmol, 85.1% yield, HRMS calculated C 88 H 103 N 11 O 15 The expected value is 1553.7635, and the measured value is 1553.7656).

[0048] Synthesis of Preparation 11 SEQ ID NO: 11 H-Alanine-O-2CTC resin complex (40.39 g, 0.5 mmol / g, 20.20 mmol) was suspended in DMF (400 mL, 10 volumes) for approximately 20 minutes, then drained. The obtained resin was washed with DMF (400 mL, 10 volumes) for 5 min twice. Standard Fmoc chemistry was used to assemble the amino acid chain. Generally speaking, 1.5 equivalents of Fmoc-amino acid and HOBt (5.51 g, 80 wt.%, 32.6 mmol, 1.6 equivalents) were dissolved in DMF (150 mL, 3.7 volumes), followed by the addition of DIEA (4.22 g, 32.7 mmol, 1.6 equivalents). The resulting solution was cooled to approximately <5°C with an ice bath, and activated by the addition of TBTU (10.39 g, 32.4 mmol, 1.6 equivalents). It was stirred at 0-5°C for approximately 5 minutes. DCM (80 mL, 2 volumes) was added to the resin, followed by the addition of activated Fmoc-amino acid solution. The resulting mixture was stirred at about ambient temperature for 2 hours.

[0049] By using 5 volume % piperidine, 1.25 volume % DBU, 1.0wt% HOBt / DMF solution (240mL, 6 volumes) to process Fmoc-amino acid resin twice (each 10min and 20min) to remove Fmoc group.Drain off-Fmoc solution, use DMF (240mL, 6 volumes) twice, MTBE (240mL, 6 volumes) twice and then use DMF (240mL, 6 volumes) twice washing resin.After completing the synthesis step, use 2-propanol (400mL, 10 volumes) twice and MTBE (400mL, each 10 volumes, 6 times) to fully wash peptide resin, then at 35 ℃ of vacuum drying, to produce the loaded resin (74.82g, 0.159mmol / g, 11.90mmol, 58.9% yield) that is not the final amino acid. The 5-nitro-1-oxo-2-nitro-4-oxo-5-nitro-1-oxo-5-nitro-1-oxo-2 ... Dissolved in DMF (50 mL, 3.7 vols), followed by the addition of DIEA (0.54 g, 4.2 mmol, 1.9 eq), Fmoc-Leu-OH (1.47 g, 4.16 mmol, 1.9 eq) and HOBt (0.704 g, 80 wt.%, 4.17 mmol, 1.9 eq) for coupling of Fmoc-Leu-OH. The resulting solution was cooled to <5°C with an ice bath and activated by the addition of TBTU (1.34 g, 4.17 mmol, 1.9 eq) and stirring at 0-5°C for 5 min. DCM (20 mL, 1.5 vols) was added to the resin, followed by the addition of the activated Fmoc-amino acid solution. The resulting mixture was stirred at approximately ambient temperature for 2 hours. The resin was washed with DMF (180 mL, 13 vols, twice), MTBE (180 mL, 13 vols, twice), and DMF (180 mL, 13 vols, twice) for 5 min each. The resin was washed with DCM (130 mL, 10 volumes, 6 times, 5 min each) before being dried under vacuum at 35°C to give the loaded resin (12.90 g, 0.203 mmol / g, 2.62 mmol, 121% yield).

[0050] A portion of the resin (7.09 g, 0.203 mmol / g, 1.44 mmol) was treated with 1% by volume TFA in DCM (70 mL, 10 volumes) three times for 10 minutes each at approximately ambient temperature, followed by washing with DCM (55 mL, 7.5 volumes). The fractions and washings were neutralized and cleaved with pyridine (3.02 g, 1.02:1 molar ratio to TFA). The fractions were combined and concentrated to dryness under vacuum at ≤35°C. Reconstitution was performed with ethanol (28 mL, 11% by volume of the combined filtrate) followed by concentration to dryness. Finally, the peptide was stirred in deionized water (105 mL, 40% by volume of the combined filtrate). The solid crude peptide precipitate was collected by filtration and washed with deionized water (4×50 mL). The solid was washed with n-heptane (3×100 mL), separated and dried under vacuum at 40° C. to afford Preparation 11 as a white powder (4.54 g, 87.6 area%, 44.4 wt% pyridine·TFA, 48.7 wt%, 0.936 mmol, 65.0% yield, HRMS calculated C 127 H 192 N 14 O 28 Expected 2361.4031, found 2361.4021). The overall yield of Preparation 11 on the resin was 71.3%.

[0051] Synthesis of Preparation 12 SEQ ID NO: 12 Fmoc-Aib-O-CTC resin complex (19.16g, 0.54mmol / g, 10.35mmol) is suspended in DMF (190mL, 10 volumes) 20min, then drain.With DMF (190mL, 10 volumes) washing gained resin 5min, then drain.Merge piperidine (77.82g), DBU (23.16g), HOBt (18.09g, 80wt%) and DMF (1800mL), to provide the solution of 5% piperidine, 1.25% DBU, 1.0% HOBt / DMF as deprotection solution.Thus remove Fmoc group by processing Fmoc-amino acid resin twice (each 10min and 20min) with deprotection solution (190mL, 10 volumes).Drain and take off-Fmoc solution, then use DMF twice, MTBE twice and again use DMF twice (190mL, each washing 10 volumes) washing resin.

[0052] Add DIEA (2.62g, 20.3mmol, 2.0 equivalents) to a solution of Fmoc-Ile-OH (7.11g, 10.1mmol, 2.0 equivalents) in DMF (85mL). Cool the resulting solution to 0-5°C, add 6-chloro-benzotriazole-1-yloxy-tripyrrolidino phosphonium hexafluorophosphate (PyClock) (11.36g, 20.06mmol, 2.0 equivalents) and dissolve completely. After standing for 3 to 5 minutes, add the activated solution to the H-Aib-O-CTC resin complex pre-swollen in DCM (30mL, 1.5 volumes). Make the reaction warming up to ambient temperature, and stir for 2 hours. As shown in the assay analysis, the unreacted material is about 18%. Wash with DMF twice, MTBE twice and DMF twice (190mL, each 10 volumes). A solution of Fmoc-Ile-OH (10.63 g, 30.08 mmol, 6 equiv) in DMF (165 mL) was added to Oxyma (50 mL, 0.6 M in DMF, 30 mmol, 6 equiv) and DIC (50 mL, 0.66 M in DMF, 33 mmol, 6.6 equiv). The mixture was stirred at approximately ambient temperature for 5 min, then added to the resin and stirred for 18 hours. A mixture of pyridine, acetic anhydride, and DMF was added to the resin and stirred for 0.5 hour. The resin was washed with DMF (5 x 140 mL, 7 volumes), more DMF (2 x 180 mL, 9 volumes), MTBE (2 x 180 mL, 9 volumes), and then DMF (2 x 180 mL, 9 volumes).

[0053] For remaining amino acid, the remaining chain assembly is implemented in order with standard Fmoc chemistry. Generally speaking, dissolve Fmoc-amino acid (2.0 equivalents), HOBt (3.42g, 80wt%, 2.0 equivalents) in DMF (85mL), then add DIEA (2.64g, 2.0 equivalents). Cool the resulting solution to 0-5 ℃ with an ice bath, and activate by adding TBTU (6.45g, 2.0 equivalents) and leaving standstill 3-5min at 0-5 ℃. Add DCM (30mL) to resin, then add activated Fmoc-amino acid solution. Gained mixture was stirred at room temperature for 2 hours. Wash the resulting resin with DMF twice, MTBE twice and again with DMF twice (190mL, each washing 10 volumes). Thus remove the Fmoc group by handling Fmoc-amino acid resin twice (each 10min and 20min) with deprotection solution (190mL, 10 volumes). After draining the de-Fmoc solution, the resin was washed twice with DMF, twice with MTBE, and twice more with DMF (190 mL, 10 volumes each wash).

[0054] The tetramer Boc-Y-Aib-E(tBu)G-OH (12.25 g, 2.0 equiv) was activated with Oxyma (30 mL, 0.6 M in DMF, 20 mmol, 2 equiv) and DIC (33 mL, 0.66 M, 22 mmol, 2.1 equiv) in DMF (50 mL) for 5 min to add the final four amino acids as a tetramer. This mixture was added to the resin and coupled for 18 hours. At the end of 18 hours, the mixture was drained and the resin was washed with DMF (190 mL, 5 min each, 5 times). More tetramer (6.21 g, 1.0 equiv) in DMF (40 mL) was added and activated with PyBOP (5.77 g, 1.1 equiv) and DIEA (3.32 g, 2.6 equiv) for 5 min and stirred for 4 hours before adding the mixture to the resin. At the end of 4 hours, the mixture was drained and washed with DMF (5 times, 190 mL, 5 min each). The resin was capped by adding a mixture of DMF (105 mL), pyridine (13.48 g, 17 eq.), and acetic anhydride (14.27 g, 14 eq.) to the resin and stirring for 1 hour. After chain assembly was complete, the peptide resin was washed with DMF five times (190 mL each), DCM six times (190 mL each), each for 5 min, and then dried under vacuum at 35° C. to give Preparation 12 resin complex (31.03 g, 0.2595 g / mmol theoretical, 8.05 mmol, 77.8% yield). A portion of Preparation 12 resin complex (15.975 g, 0.2595 mmol / g, 4.146 mmol) was treated with 1% by volume TFA in DCM (160 mL, 10 volumes) three times for 10 minutes each at ambient temperature, followed by washing with DCM (120 mL, 7.5 volumes). The cleavage fractions and washings were neutralized with pyridine (4.74 g, 0.94:1 molar ratio to TFA). The fractions were combined and concentrated to dryness under vacuum at ≤35°C. Reconstitution was performed with ethanol (30 mL, 5% by volume of the combined filtrate) followed by concentration to dryness. The peptide was mechanically stirred in deionized water (242 mL, 40% by volume of the combined filtrate) for 10 minutes. The solid crude peptide was collected by filtration and washed with deionized water (4×100 mL). The solid was washed with n-heptane (4 x 100 mL), separated, and dried under vacuum at 35 °C to afford Preparation 12 as a white powder (9.38 g, 82.2 area%, 0.2 wt% pyridine·TFA, 82.1 wt%, 3.85 mmol, 92.8% yield, HRMS calculated C 103 H 165 N 13 O 26 The expected value is 2000.1989, and the measured value is 2000.1968).

[0055] Synthesis of Preparation 13 SEQ ID NO: 13 To a flask under N2 was added Preparation 9 (2.887 g, 70.2 wt%, 1.30 mmol), Preparation 10 (3.576 g, 57.4 wt%, 2.13 mmol, 1.63 equiv), DMSO (18.1 g, 16.4 mL), DMF (15.8 g, 16.7 mL) and DIEA (655 mg, 5.07 mmol, 3.89 equiv) with stirring until a golden solution was obtained. The solution was cooled in ice water before adding PyBOP (1.414 g, 2.72 mmol, 2.08 equiv). The ice bath was removed and the mixture was allowed to warm to ambient temperature. The reaction was monitored for approximately 5 hours to ensure sufficient conversion. An aliquot of diethylamine (2.116 g, 28.9 mmol, 22.2 equiv) was added to the ambient temperature reaction mixture. The mixture was stirred for approximately one hour to provide a conversion of approximately >99% to Preparation 13. The product was precipitated by adding a mixture containing saturated aqueous NaHCO3 (50 mL) and deionized water (50 mL) at <4°C to the reaction mixture. The mixture was stirred for at least about 15 minutes under cold conditions. The muddy white slurry was filtered. The wet cake was washed with deionized water (3×50 mL) followed by MTBE (6×50 mL) and dried at 40°C in a vacuum purged with N2 for about 62 hours. This method gave Preparation 13 (4.45 g, 60.4 area%, 16.4 area% dibenzofulvene, 1.18 mmol, 90.5% yield, HRMS calculated C 119 H 169 N 21 O 24 Expected value 2276.2649, measured value 2276.2550), light yellow solid.

[0056] Synthesis of Preparation 14 SEQ ID NO: 14 Under N2, an aliquot of Preparation 11 (3.012 g, 48.7 wt%, 0.621 mmol, 1.00 equiv) was added to a flask containing Preparation 13 (3.951 g, 60.4 wt%, 1.05 mmol, 1.69 equiv), DMSO (9.8 g, 8.9 mL), DMF (52.0 g, 55.0 mL), and DIEA (372 mg, 2.88 mmol, 4.63 equiv). The mixture was stirred until a golden solution was obtained. Ice water was added to cool the mixture to <10°C. An aliquot of PyBOP (742 mg, 1.42 mmol, 2.30 equiv) was added to the mixture. The ice bath was removed and the mixture was allowed to warm to approximately ambient temperature. The reaction was monitored for approximately 22 hours to produce Preparation 14. This resulted in a conversion of approximately >96%. When the temperature is <10°C, piperidine (530 mg, 6.22 mmol, 10.0 equivalents) is added to the cooled reaction mixture. The mixture is stirred at ambient temperature for about 2 hours to provide a conversion rate of> about 99% to Preparation 14. The reaction mixture is added to another flask containing <4°C 0.5N HCl aqueous solution (12.72 g, 6.23 mmol, 10.0 equivalents) and deionized water (16.71 g) to provide a precipitate of Preparation 14. The cold slurry is stirred for about 15 minutes, and the white slurry is filtered. The wet cake is washed with deionized water (2 × 30 mL), saturated NaHCO3 aqueous solution (2 × 30 mL), deionized water (3 × 30 mL) and MTBE (4 × 45 mL) and dried at 40°C in a vacuum purged with N2 for about 17 hours. The product Preparation 14 (5.418 g, 48.9 area%, 0.603 mmol, 97.0% yield, HRMS calculated C 231 H 349 N 35 O 49 The expected value is 4397.5893, the measured value is 4397.6057), and it is a white powder.

[0057] Synthesis of Preparation 15 SEQ ID NO: 15 An aliquot of Preparation 12 (671 mg, 82.1 wt%, 0.275 mmol, 1.23 equiv) was added to a flask under N2. Preparation 14 (2.009 g, 48.9 area%, 10.7 area% isomers, 0.223 mmol, 1.00 equiv), DMSO (11.1 g, 10.0 mL), DMF (19.0 g, 20.1 mL), and DIEA (76 mg, 0.588 mmol, 2.63 equiv) were added to the flask with stirring to give a golden solution. An aliquot of 0.6 M HOAt (619 mg, 0.384 mmol, 1.72 equiv) was added before cooling to -5°C. A sample of PyClock (220 mg, 0.397 mmol, 1.78 equiv) was added. The mixture was allowed to warm to approximately ambient temperature to provide approximately 84% conversion to Preparation 15. The product was isolated by adding the reaction mixture to ice-cold deionized water (548 mL) over 10 min to obtain a precipitate of the product. The reaction flask was rinsed with DMF (5 mL) and added to the slurry. The slurry was stirred for about 15 minutes, allowed to warm to about ambient temperature and filtered. The wet cake was washed with deionized water (3 x 80 mL), and the white waxy solid was dried at 35 ° C in a vacuum for 3.5 days to obtain Preparation 15, (2.506 g, 41.6 area%, 0.163 mmol, 73.1% yield, HRMS calculated C 334 H 512 N 48 O 74 The expected value is 6379.7777, the measured value is 6379.8652), and it is a white powder.

[0058] Example 2 Synthesis of Example 2 SEQ ID NO: 1 Under N2, a sample of TFA (19.656g, 13.03mL) was added to a flask containing DCM (815mg, 0.62mL), DTT (434mg) and TIPS (362mg, 0.47mL). Before adding water (468mg, 0.47mL), the mixture was cooled in ice water. At 2°C, a sample of preparation 15 (1016mg, 39.0 area%, 0.0620mmol) was added to the mixture to provide a solution. The mixture was warmed to approximately ambient temperature and stirred for approximately 2 hours. The reaction mixture was added to -15°C MTBE (150mL) and the reactor was rinsed with MTBE (3mL). The slurry was centrifuged after approximately 10 minutes and the supernatant was decanted. The wet cake was re-slurried in MTBE (3 × 50mL), centrifuged at each washing, and the supernatant was decanted. The wet cake was dried at 35 °C in vacuo to give Example 2 (784 mg, 26.5 area%, 0.0432 mmol, 69.7% yield, HRMS calculated C 225 H 348 N 48 O 68 The expected value is 4810.5249, the measured value is 4810.5642), which is a white solid.

[0059] Synthesis of Preparation 16 SEQ ID NO: 16 The synthesis used Fmoc-Gly-OH-2-chlorotrityl resin with a loading of 0.61 mmol / g. The general SPPS procedure was essentially the same as described herein. Preparation 16 was obtained from soft cleavage of the peptide on the resin using methods known to those skilled in the art, such as those described herein. The concentrated material was reconstituted with ethanol (5% volume of the combined filtrate) and concentrated to dryness. The peptide was triturated in water (40% volume of the combined filtrate) with stirring. The solid was isolated and dried under vacuum at 40° C. to constant weight to produce 5.24 g (99%) of Preparation 16 as a white powder.

[0060] Synthesis of Preparation 18 SEQ ID NO: 17 The synthesis used Fmoc-Ala-OH-2-chlorotrityl resin with a loading of 0.50 mmol / g. The general SPPS protocol used was essentially the same as described herein with the following modifications:

[0061] Preparation 18 Soft Lysis: The peptide intermediate sample on 42.13g resin is placed in a flask and treated with 10 volumes (400mL) of 1% TFA / DCM 3 times for 10min each time, then washed with DCM. Each treatment is quenched by adding 4.4mL pyridine. The resulting solution is combined and concentrated in a vacuum. Reconstructed with ethanol (25mL), then concentrated to dryness to obtain 56.6g foamy semi-solid. Add 400mL volume of water 10 times to provide slurry. The slurry is filtered and washed with water. The solid is separated and dried to constant weight at 40°C under vacuum to produce 23.3g preparation 18, which is a white powder.

[0062] Synthesis of Preparation 17 ((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-tetraazatricarboxane-53-ol) Preparation 6 (80 g, 92 mmol), DIEA (17.53 mL, 101 mmol), TSTU (30.3 g, 101 mmol) and acetonitrile (1 L) were added to a vessel and stirred at 23° C. for 17 hours. The solution was concentrated and the resulting orange residue was redissolved in EtOAC (1.6 L) and then washed with 0.1 M HCl (2×1 L). The organic layer was washed with water (2×1 L) and then dried over MgSO , filtered and concentrated in vacuo to leave an orange oil (83 g). A second batch was run on the same scale and combined to provide 123 g of crude oil. The intermediate ester (123 g, 110 g active, 113 mmol) was dissolved in EtOH (700 mL) and Fmoc-lysine (45.9 g, 125 mmol) and DIEA (21.70 mL, 125 mmol) were added and the reaction was stirred for 17 hours. After the reaction was completed, EtOH was removed in vacuo and an orange oil (201g) was left. The residue was dissolved in EtOAc (1.1L) and washed with 0.1M HCl solution (3 × 400mL), then with NaHCO The aqueous solution (400mL) was washed. The layers were separated and the organic layer was washed with saturated sodium chloride solution (1 × 400mL). Organic matter was concentrated to produce an orange oil (~190g). Acetone (400ml) was added and the resulting suspension was filtered to remove inorganic matter. The mixture was concentrated and purified by normal phase chromatography (1.1kg silica gel prepared with 60 / 40 heptane / acetone) and eluted with an eluent of increasing polarity (collecting~3L fractions). The component of at least 95HPLC area % was merged and concentrated to provide a viscous yellow oil (70g) for preparation 17.

[0063] Synthesis of Preparation 19A SEQ ID NO: 18 The synthesis used Fmoc-Leu-OH 2-chlorotrityl resin with a loading of 0.65 mmol / g. The general SPPS protocol was used with the following modifications:

[0064] Pseudoproline-derivatized Preparation 19A can be processed as Example 3 in a similar manner to Preparation 19B described herein.

[0065] Synthesis of Preparation 19B SEQ ID NO: 19 The synthesis used Fmoc-Leu-OH-2-chlorotrityl resin at a loading of 0.65 mmol / g. Preparation 19B was prepared using essentially the same SPPS procedure as described herein.

[0066] Preparation 19B soft lysis: Preparation 19B was prepared using soft cleavage of resin-bound 19B essentially as described herein using methods known to those skilled in the art. See, for example, the method for Preparation 18. The resulting solid was isolated and dried under vacuum at 30-40° C. to constant weight to yield 2.94 g of product as a light yellow powder.

[0067] Synthesis of Preparation 20 SEQ ID NO: 20 To a solution of Preparation 1 (4.25 g, 4.166 mmol) and Preparation 16 (5.00 g, 3.340 mmol) in DMSO / DMF (1:1, 200 mL) was added PyBOP (2.60 g, 5.00 mmol) and DIEA (1.75 mL, 10.0 mmol) at ambient temperature. The solution was stirred for 18 hours and then quenched by adding an excess of diethylamine (10.0 mL). The quenched solution was stirred for 2 hours and then slowly added to a saturated sodium bicarbonate / water (1:1, 300 mL) solution at 0°C. The resulting precipitate was stirred for 10 minutes and then collected by filtration. The filtrate was washed sequentially with water (3×150 mL) followed by methyl tert-butyl ether (3×150 mL). The solid was dried under vacuum at 40°C to provide Preparation 20 as a white solid (5.30 g, 69% yield, HRMS calculated C 119 H 169 N 21 O 24The expected value is 2276.2649, and the measured value is 2276.2652).

[0068] Synthesis of Preparation 21 SEQ ID NO: 21 To a solution of Preparation 20 (1.00 g, 0.44 mmol) and Preparation 18 (0.90 g, 0.40 mmol) in DMSO / DMF (1:1, 20 mL) was added PyBOP (314 mg, 0.30 mmol) and DIEA (0.21 mL, 1.20 mmol) at ambient temperature. The solution was stirred for 18 hours and then quenched with piperidine (0.79 mL, 4.00 mmol). The quenched solution was stirred for 2 hours and then cooled to 0°C and quenched with a dilute HCl solution (50 mL). The resulting slurry was stirred for 10 minutes and the solid was collected by filtration. The filtrate was washed sequentially with saturated aqueous sodium bicarbonate solution (2×50 mL), water (3×50 mL), and then methyl tert-butyl ether (3×50 mL). The solid was dried under vacuum at 40°C for 18 hours to provide Preparation 21 as a white solid (1.80 g, 106% yield, HRMS calculated C 225 H 338 N 34 O 48 The expected value is 4284.5053, and the measured value is 4284.5062).

[0069] Synthesis of Preparation 22 SEQ ID NO: 22 To a solution of Preparation 21 (214 mg, 0.05 mmol) and Preparation 19B (116 mg, 0.055 mmol) in DMSO / DMF (1:1, 3 mL) was added PyBOP (57 mg, 0.11 mmol) and DIEA (58 μL, 0.33 mmol) at ambient temperature. The solution was stirred for 18 hours and then quenched with a 1:1 mixture of saturated aqueous sodium bicarbonate solution and water (10 mL). The resulting slurry was stirred for 10 minutes, and the resulting solid was collected by filtration. The solid was washed with water (3×10 mL) and dried under vacuum at 40° C. to provide Preparation 22 (285 mg, 89% yield, HRMS calculated C 334 H 512 N 48 O 74 The expected value is 6379.7777, and the measured value is 6379.7730).

[0070] Example 3 Synthesis of Example 3 SEQ ID NO: 1 A solution of TFA (2.3 mL), water (0.1 mL), triisopropylsilane (0.1 mL) and DTT (75 mg) was cooled to 0 ° C. Preparation 22 (100 mg, 0.015 mmol) was added to the solution and the reaction mixture was warmed to ambient temperature and stirred for 2 hours. The resulting mixture was poured into a pre-cooled (-20 ° C) solution of methyl tert-butyl ether (25 mL). The resulting precipitate was kept at -20 ° C for 15 minutes, kept for 15 minutes, the slurry was centrifuged, and washed with methyl tert-butyl ether (2×25 mL). The solid was dried under vacuum at 35 ° C for 18 hours to obtain Example 3 as a white solid (71 mg, 93% yield, HRMS calculated C 225 H 348 N 46 O 68 The expected value is 4810.5249, and the measured value is 4810.5036).

[0071] Step 1 Step 1: Prepare a feed solution of Preparation 25 (1.05 equiv) in 5 volumes of DMSO / ACN (90:10 v / v). Prepare a second feed solution of Preparation 26 in 20 volumes of DMSO / ACN (90:10 v / v). Prepare a third feed solution of PyOxim (1.5 equiv) in 3 volumes of ACN. Prepare a fourth stream of DIEA (4 equiv) in ACN. Pump the first three streams into a mixer, and mix the DIEA at the mixer outlet. The mixture is pumped through another mixer and passed through a plug flow reactor with a residence time of 2 hours in a 20°C constant temperature bath. At the reactor outlet, acetic acid may be added to consume any remaining PyOxim (1-cyano-2-ethoxy-2-oxoethylideneaminooxy-tripyrrolidinophosphonium hexafluorophosphate). After >2 hours, add neat diethylamine (10 equiv) and mix with the mixer. This stream entered a second plug flow reactor with a residence time of 1 hour in a thermostatic bath at 20° C. The product solution of Preparation 27 was collected and passed through nanofiltration with a 70 / 30 DMSO / ACN solution to remove 10-20 diavolumes of reagents.

[0072] A feed solution of Preparation 25 (2.40 kg, 96.7 wt%, 2.274 mol) was prepared by dissolving the solid in DMSO (13.88 kg, 12.62 L) and diluting the solution with ACN (1.09 kg, 1.39 L) to produce a solution of Preparation 25 in 90:10 DMSO:ACN volume / volume (114.3 mg / mL, 0.112 M). A second feed solution of Preparation 26 (SEQ ID NO: 26, 2.79 kg, 98.6 wt.%, 1.836 mol) was prepared by dissolving the solid in DMSO (54.8 kg, 49.8 L) and diluting the solution with ACN (4.3 kg, 5.47 L) to produce a solution of Preparation 26 in 90:10 DMSO:ACN volume / volume (45.5 mg / mL, 0.030 M). A third feed solution was prepared from PyOxim (4.5 kg, 8.53 mol) in ACN (47.33 kg, 60.22 L) to yield a 0.132 M solution. DIEA was added neat as a liquid. At 20° C., a solution of Preparation 25 (14.91 L, 1.704 kg, 1.670 mol, 0.95 eq., 5.9 g / min) and Preparation 26 (58.46 L, 2.660 kg, 1.750 mol, 1.00 eq., 22.5 g / min) and PyOxim (1.4 eq., 5.4 g / min) were pumped into a mixer and mixed with neat DIEA (4.0 eq., 0.446 mL / min). The mixture was pumped through another mixer and passed through a plug flow reactor with a residence time of 3 hours in a 20° C. constant temperature bath and collected over 42.9 hours to yield 88.6 kg of product solution.

[0073] Nanofiltration is a membrane-based filtration method used to separate chemical substances based on their size and molecular weight differences. The product solution of Preparation 27 contains reagents (diethylamine, PyOxim, DIEA, etc.) and unwanted by-products (e.g., dibenzofulvene) that need to be removed before proceeding to the next step. Nanofiltration is used to remove unwanted substances (molecular weight <500 Da).

[0074] The product solution of Preparation 27 is charged to the NF feed tank and pumped in a circulation loop through a heat exchanger and a nanofiltration unit containing a suitable membrane (ceramic or polymer) to achieve the desired separation. Unwanted substances are removed in the permeate and collected separately or discarded to waste. To maintain a constant volume in the NF tank, fresh solvent, i.e., 70:30 v / v DMSO / ACN, is continuously pumped in to match the resulting permeation rate. The product solution of Preparation 27 purified by nanofiltration proceeds directly to Step 2.

[0075] To the reactor was added a solution of Fmoc-protected Preparation 27 in DMSO / ACN (88.6 kg) and diethylamine (1.34 kg). The mixture was stirred at 20° C. for 2 hours to give Preparation 27 (87.6 L, 38.45 mg / mL, 3.37 kg, 1.48 mol). The product solution of Preparation 27 was charged to a nanofiltration feed tank and then pumped through a heat exchanger and a nanofiltration unit containing a suitable membrane (ceramic or polymer) in a circulation loop to achieve the desired separation. Unwanted substances were removed on the permeate side and collected separately or discarded as waste. This operation was continued until sufficient removal of unwanted impurities was achieved. In order to maintain a constant volume in the nanofiltration tank, fresh solvent, i.e., 70:30 volume / volume DMSO / ACN, was continuously pumped in to match the resulting permeability. This gave Preparation 27 in DMSO / ACN (72.4 L, 40.8 mg / mL, 2.95 kg, 1.30 mol, 78.1% yield by coupling, Fmoc removal and nanofiltration).

[0076] Step 1 Example - Analyze the results.

[0077] HPLC confirmed the conversion of Preparation 25 and Preparation 26 to form Preparation 27. The analytical method used a phenylhexyl stationary phase column (2.1 mm ID x 150 mm x 1.7 micron particle size) at 65°C with a 2-98% B gradient over 12 minutes of 0.1% TFA in water and acetonitrile. The material was detected by UV at 214 nm.

[0078] Table A.1 shows the high-resolution mass spectrometric data collected for the product of the Step 1 coupling reaction (Fmoc-protected Preparation 27) and the product of the Step 1 deprotection reaction (Preparation 27). Mass accuracy is a measure used to confirm that the measured species matches the predicted species.

[0079]

[0080] Table A.1 Confirmation of measured mass accuracy of Fmoc-protected Preparation 27 and Preparation 27 calculated using high resolution mass spectrometry data.

[0081] Step 2 Scheme A.2 Synthesis of Preparation 27 (SEQ ID NO: 27) and Preparation 28 (SEQ ID NO: 28) from fragments Preparation 27 (SEQ ID NO: 27) and Preparation 28 (SEQ ID NO: 28) 29 (SEQ ID NO: 29).

[0082]

[0114] Step 2: A feed solution of Preparation 28 (1.15 eq) was prepared in 10 volumes of DMSO / ACN (90:10 v / v). A second feed solution was prepared which was PyOxim (2 eq) in 1 volume of ACN. A third stream was prepared which was DIEA (3 eq) in ACN (5 wt% solution). The solution of Preparation 27 from Step 1 and the streams of Preparation 28 and PyOxim were pumped into a mixer and DIEA was mixed at the outlet of the mixer, the mixture was pumped through another mixer and passed through a plug flow reactor with a residence time of 2 hours in a constant temperature bath at 20°C. At the reactor outlet, acetic acid may be added to consume residual PyOxim. After >2 hours, neat diethylamine (10 eq) was added and mixed through a mixer. This stream entered a second plug flow reactor with a residence time of 1 hour in a constant temperature bath at 20°C. The product solution of Preparation 29 was collected and passed by nanofiltration with DMF solution as the diafiltrant to remove 10-20 diafiltration volumes of reagents.

[0083] A feed solution of Preparation 28 (4.68 kg, 98.9 wt.%, 2.058 mol) was prepared by dissolving the solid in DMSO (41.75 kg, 37.95 L) and diluting the solution with ACN (3.3 kg, 4.20 L) to produce a solution of Preparation 28 (94.7 mg / mL, 0.0421 M) in 90:10 DMSO:ACN volume / volume. A second feed solution was prepared of PyOxim (3.0 kg, 5.69 mol) in ACN (11.81 kg, 15.03 L) to produce a 0.327 M solution. DIEA was added as a neat liquid. A solution of Preparation 27 from Step 1 (73.86 L, 41.2 mg / mL, 3.04 kg, 1.336 mol, 0.0181 M, 1.0 equiv, 29.9 g / min) and Preparation 28 (1.3 equiv, 17.7 g / min) along with PyOxim (2.1 equiv, 2.9 g / min) were pumped into a mixer and the stream was adjusted to 20° C. at the mixer outlet and combined with neat DIEA (4.0 equiv, 0.374 mL / min). The mixture was pumped through another mixer and through a plug flow reactor with a residence time of 3 hours in a bath at 20° C. and collected over 43.2 hours to give 129.35 kg of Preparation 29 product solution.

[0084] The nanofiltration process was essentially as described above, using the product solution of Preparation 29 instead of the product solution of Preparation 27.

[0085] The nanofiltration method using a solution of Fmoc-protected Preparation 29 in DMSO / ACN was performed essentially as described herein. Fmoc-protected Preparation 29 (129.35 kg) and diethylamine (2.0 kg) were added to a reactor for nanofiltration. The nanofiltration method produced Preparation 29 in DMF (98.85 L, 42.24 mg / mL, 4.18 kg, 0.974 mol, 73.1% yield via coupling, Fmoc removal, and nanofiltration).

[0086] HPLC confirmed the synthesis of Preparation 29 from Preparation 28 and Preparation 27. The analytical method used a C4 stationary phase column (2.1 mm ID x 150 mm x 1.7 micron particle size) at 65° C. with a 25-98% B gradient over 12 minutes of 0.1% TFA in water and acetonitrile. UV detection at 214 nm was used for this material.

[0087] The gradient was 25-98% B with 0.1% TFA in water and acetonitrile over 12 minutes.The material was detected by UV at 214 nm.

[0088] Table A.3 shows the high-resolution mass spectrometric data collected for the Step 2 coupling reaction product (Fmoc-protected Preparation 29) and the Step 2 deprotection reaction product (Preparation 29). The mass accuracy confirms the measured material product. Monoisotopic masses of neutral material.

[0089] Table A.3. Measured Fmoc-protected Preparation 29 and Preparation 29 confirmed by mass accuracy calculated using high resolution mass spectrometry data.

[0090] Step 3 Scheme A.3 Synthesis of Preparation 30 (SEQ ID NO: 30) and Preparation 29 (SEQ ID NO: 29) from fragments 31 (SEQ ID NO: 31).

[0091] Step 3 Batch method description: To a nanofiltered DMF solution of Preparation 29 (2.249 g, 46.5 mg / g, 104.6 bmg, 0.0244 mmol) and Preparation 30 (71.5 mg, 90.6 area %, 0.0306 mmol) in DMF (0.3068 g, 0.325 mL) at -5 °C was added 5.0 wt% DIEA in DMF (114.0 mg, 5.70 mg DIEA, 0.0441 mmol, 1.8 equiv) and 10.1 wt% (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) in DMF (154.9 mg, 15.59 mg HATU, 0.0410 mmol, 1.7 equiv). The solution was stirred at -5°C for 4 hours and then quenched with 5 wt% aqueous sodium bicarbonate solution (5.295 g, 4.8 mL) added over 15 minutes at ambient temperature. The resulting slurry was stirred at 0°C for 15 minutes, and the resulting solid was collected by filtration. The solid was washed with water (4 x 2 mL) and then with MTBE (4 x 2 mL). The sticky solid was dried under vacuum at 35°C to give Preparation 31 (219.7 mg, 47.6 area %, 0.0164 mmol, 67.1% yield).

[0092] A feed solution of Preparation 30 was prepared in 10 volumes of DMF. A second feed solution was prepared as 10 wt% HATU (1.8 eq) in ACN. A third stream was prepared as DIEA (2.5 eq) in DMF (5 wt% solution). The solution of Preparation 29 from Step 2, along with the Preparation 30 and DIEA streams, were pumped into a mixer, and the streams were cooled at the outlet of the mixer and combined with the cooled HATU solution. The mixture was pumped through another mixer and through a plug flow reactor with a residence time of 3 hours in a -5°C constant temperature bath and collected. A brine / bicarbonate solution with a 19% salt loading by weight was prepared (17 wt% sodium chloride in water, 0.5 wt% sodium bicarbonate in water). The product solution of Preparation 31 in DMF was then pumped into a mixed product mixed suspension reactor (MSMPR) along with the brine solution to form a precipitate. The τ (tau) in the mixed product mixed suspension reactor was 1 hour. The second MSMPR was run at cooler conditions for 1 hour of τ and the slurry was then fed intermittently to the filter. The slurry was washed with water and dried under vacuum at 35°C.

[0093] A feed solution of Preparation 30 (9.42 kg, 83.6 wt%, 3.72 mol) was prepared in DMF (54.12 kg) to produce a 0.0563 M feed. A second feed solution of HATU (1.15 kg, 3.02 mol) in ACN (10.4 kg) was prepared to produce a 0.215 M feed. DIEA was added as a neat liquid. A solution of Preparation 29 from Step 2 (98.85 L, 42.24 mg / mL, 4.18 kg, 0.975 mol, 0.0099 M, 1.0 eq, 40.2 g / min) and Preparation 30 (1.3 eq, 9.2 g / min) were pumped into the mixer along with a stream of DIEA (2.1 eq, 0.152 mL / min). At the mixer outlet, the stream was cooled to 0°C and combined with cooled HATU solution (2.0 equivalents, 3.2 g / min) at 0°C. The mixture was pumped through another mixer and through a plug flow reactor with a residence time of 4 hours in a bath at 0°C and collected over 42 hours to obtain 131.8 kg of product solution. The product solution was precipitated in two portions. 17 wt% NaCl aqueous solution / 0.5 wt% NaHCO3 aqueous solution (29 kg) was combined with DMF (13.2 kg) in an inert reactor and cooled to no more than 20°C. The product solution in DMF (66.6 kg) and 17 wt% NaCl aqueous solution / 0.5 wt% NaHCO3 aqueous solution (34.4 kg) were then added to the reactor over 1 hour, maintained at 20°C, to obtain precipitation of product Preparation 31. The slurry was cooled to 5°C over 1 hour before water was added in two portions (total 32.2 kg). The 5°C slurry was stirred for 0.5 hours before filtering the slurry. The wet cake was reslurried in water (63.9 kg) for 0.5 hours and filtered. The second portion of the product solution precipitated in a comparable manner. 17 wt% NaCl aqueous solution / 0.5 wt% NaHCO aqueous solution (29 kg) and DMF (13.55 kg) were combined in a reactor and cooled to no more than 20° C. The second portion of the product solution in DMF (65.2 kg) was co-added to the reactor with 17 wt% NaCl aqueous solution / 0.5 wt% NaHCO aqueous solution (36.8 kg) over 1 hour, maintained at 20° C., to obtain a precipitate of product Preparation 31. The slurry was cooled to 5° C. over 1.25 hours, and then water was added in two portions (32.1 kg in total). The slurry was stirred for 0.5 hours, and then the slurry was filtered on the first wet cake. The combined wet cake was reslurried twice with water (64 kg each), each for 0.5 hours, and filtered. Two replacement water washes (64 kg each) were subsequently performed.The combined washed wet cakes were purged with N2 and then dried under vacuum at 38°C until KF < 4 wt% to afford Preparation 31 (10.34 kg, assumed 60% potency, 0.972 mol, assumed 100% yield).

[0094] Example 3 Tirzepatide (SEQ ID NO: 1) A solution of TFA (2.3 mL), water (0.1 mL), triisopropylsilane (TIPS, 0.1 mL) and dithiothreitol (DTT, 75 mg) was cooled to 0 ° C. Preparation 31 (100 mg, 0.015 mmol) was added to the solution, and the reaction mixture was warmed to ambient temperature and stirred for 2 hours. The resulting mixture was poured into a pre-cooled (-20 ° C) MTBE solution (25 mL). The resulting precipitate was kept at -20 ° C for 15 minutes, kept for 15 minutes, and the slurry was centrifuged and washed with MTBE (2×25 mL). The solid was dried under vacuum at 35 ° C for 18 hours to obtain Example 3 as a white solid (71 mg, 93% yield, HRMS calculated C 225 H 348 N 46 O 68 The expected value is 4810.5249, and the measured value is 4810.5036).

[0095] To an inerting reactor at 15°C was added DCM (27.3 kg, 20.6 L), water (4.1 kg), Preparation 31 (10.34 kg, assuming 60% potency, 0.972 mol), and DTT (3.10 kg). In a separate inerting reactor were added TFA (154.1 kg, 103.4 L) and TIPS (3.2 kg, 4.2 L). The TFA / TIPS solution was added to the slurry of Preparation 31, DCM, water, and DTT over 0.25 hours to form a colorless solution, which was warmed and held at 20°C for 3 hours. After 3 hours at 20°C, the reactor was cooled to -10°C. In a separate reactor, MTBE (382.4 kg, 516.8 L) was added and cooled to -20°C. A portion of this cold MTBE (91.8 kg, 124.1 L) was added to the cold reaction solution over 2 hours, maintaining between -5°C and -18°C. The remaining cold MTBE (294.3 kg, 397.7 L) was added over 1.5 hours, maintaining at -5°C to -18°C, to give the precipitate of Example 3. The slurry was adjusted to 0°C and held for >0.5 hour, then filtered in three portions. The combined wet cake was reslurried twice with MTBE (114.7 kg, 155 L) and filtered before the final MTBE displacement wash (114.7 kg, 155 L). The wet cake was dried at 28°C until <4.5 wt% MTBE was measured. This gave Example 3 (7.77 kg, 46.8 wt.%, 0.755 mol, 77.7% yield).

[0096] Example 4A (linear SPPS) Tirzepatide (SEQ ID NO: 1) Fmoc Sieber resin (17kg, 0.76mmol / g) was added to the reactor. The resin was swollen with DMF and stirred for 2 hours, and then the DMF was filtered out from the resin. The resin was then washed twice with DMF. The Fmoc-protected resin was then deprotected using 20% ​​PIP / NMP. After the last PIP / NMP treatment, a sample was taken to verify Fmoc removal, and UV analysis confirmed that >99% Fmoc was removed (IPC target <1% Fmoc remained). After the final 20% w / w PIP / NMP treatment, the resin bed was washed multiple times with DMF. Then, for each amino acid coupling and deprotection, the following general conditions were used to construct the peptide backbone.

[0097] Fmoc Deprotection: The resin in the peptide reactor was treated with three or four loads of a 20% v / v PIP / NMP solution. Each treatment was stirred on the resin for 30 minutes and then filtered to complete the removal of the Fmoc protecting group. After the final 20% v / v PIP / NMP treatment, the resin bed was washed a minimum of six times with DMF using the pre-specified volumetric loadings.

[0098] Amino Acid Activation: A previously prepared 12% w / w Oxyma Pure / NMP solution was added to the reactor. The selected Fmoc amino acid was then added. The mixture was stirred at 20 ± 5°C until the Fmoc amino acid was completely dissolved. Prior to activation, the Fmoc-AA / Oxyma Pure / NMP solution was cooled to 15 ± 3°C to ensure control of the slightly exothermic activation reaction and to maintain the resulting solution temperature within the specified 20 ± 5°C range. The amino acid solution was then activated by the addition of DIC. The activated ester solution was then stirred for 20–30 minutes before transferring the solution to the reactor containing the peptide intermediate on the resin.

[0099] Coupling: After the pre-activation step is completed, the activated ester solution is transferred to the reactor containing the deprotected peptide on the resin to initiate the coupling reaction. The peptide coupling reaction is stirred at 20±5°C for at least 4 hours. After the required stirring time, the resin slurry is sampled to complete the coupling (IPC). Sampling is repeated at specific time intervals as needed until qualified IPC results are obtained. If necessary, re-coupling operation is performed. When the coupling is complete, the contents of the peptide reactor solution are filtered, and the peptide intermediate on the resin is then washed several times with DMF to prepare for the next coupling.

[0100] Coupling of Ile (12) with Aib (13): The coupling of Fmoc-Ile (12) with Aib (13) was performed using the symmetrical anhydride method, using six equivalents of Fmoc-AA and three equivalents of DIC. For this sequence, the activation time was extended to 40-60 min to ensure the formation of the activated symmetrical anhydride species. An extended coupling stirring time (18 h) was required to achieve reaction completion (<1% uncoupling), as determined by HPLC analysis.

[0101] Lys(20)ivDe deprotection (Preparation 23): Selective deprotection of the fully protected 39 amino acid Lys(20)ivDde group on the resin Boc-Tyr(1)-Ser(39) peptide backbone was performed. Deprotection was achieved using an 8% w / w solution of hydrazine hydrate in DMF with stirring at ambient temperature for 4 hours. The deprotection reaction was monitored by HPLC with a target IPC limit of <1% for the remaining Lys(ivDde) ​​component after deprotection. The resulting peptide fragment (Preparation 23) was repeatedly washed (8x) with DMF to completely remove residual hydrazine. The fully constructed Preparation 23 fragment was washed four times with IPA and then dried at ≤40°C until LOD ≤1% was achieved. Preparation 23 was packaged and refrigerated (-20°C) prior to coupling with Preparation 6.

[0102] Coupling of Preparation 6 with Preparation 23: Preparation 6 (1.5 eq) and PyBOP (1.5 eq) were added to a reactor as solids, followed by the addition of DMF and the mixture was stirred until dissolution occurred. Trimethylpyridine was then added to initiate the formation of the active ester species. The activated ester solution was stirred for 60 min and then transferred to the reactor containing the Preparation 23 intermediate. The reaction slurry was stirred at 25°C for 18 hours. The slurry was sampled to complete the coupling (IPC) and repeated at specific time intervals as necessary to achieve a qualified IPC (<1% Preparation 23) result. After the coupling was completed, the solution contents were filtered and discarded. The fully constructed Preparation 24 intermediate was washed several times with DMF and then with IPA. Preparation 24 was dried at ≤40°C until LOD ≤1% was reached. Preparation 24 was packaged and refrigerated (-20°C) before being cleaved from the resin.

[0103] Resin cleavage and crude product isolation from Example 4A: Prepare a cleavage mixture consisting of trifluoroacetic acid (TFA), triisopropylsilane (TIPS), dimercaptoethanol (DTT), DCM, and water. Cool the cleavage mixture to 15 ± 5°C. The reagents are as shown in the following table:

[0104] Preparation 24 was added to the reactor, followed by the cleavage mixture. The mixture was stirred and maintained at 23°C for 3 hours. The mixture was filtered, and the spent resin was washed with DCM. The DCM wash filtrate was combined with the bulk deprotection solution, and the contents were cooled to ≤-10°C. MTBE was cooled to ≤-13°C. Cold MTBE was then added to the cold filtrate in two portions. The MTBE feed rate was controlled to maintain the internal temperature of the crude solution at ≤5°C. The initial MTBE charge comprised ~45% of the total MTBE charge. A soft precipitate formed near the end of the MTBE addition but readily redissolved into solution. The precipitate solution was then recooled to an internal temperature of -15±5°C. The second MTBE addition was performed at a feed rate approximately 5-10 times the initial MTBE feed rate and comprised ~55% of the total MTBE charge. During this addition, the internal temperature of the precipitate slurry was maintained at ≤0°C. The resulting slurry was aged at -8±3°C for a minimum of 6 hours, then warmed to 0±3°C and aged for an additional 2 hours before separation.

[0105] The cold crude peptide slurry was filtered and the resulting wet cake was washed with MTBE. The crude wet cake of Example 4A was then dried to an IPC target LOD value of <1%. The crude product of Example 4A was packaged and stored. The crude intermediate was refrigerated (-20°C) until purification. Overall, 45.39 kg of crude Example 4A was produced with an HPLC area percentage purity of 45 wt% and 64%. Contained yield based on Sieber resin = 47%.

[0106] Example 4A Purification: Mobile phase: Mobile phase A (MPA) 90% water, 0.1% TFA, and 10% ACN Mobile phase B (MPB) 10% water, 0.1% TFA and 90% ACN Reverse phase purification 1 (RP1): The crude product of Example 4A was dissolved in 90wt% MPA and 10wt% MPB. The solution was stirred for at least 7 hours to complete the tryptophan decarboxylation reaction. The aged crude product solution was filtered and loaded onto a pre-equilibrated Kromasil 100-10-C8 packed column. The column was washed with a mixture of A (90% water, 0.1% TFA and 10% ACN) and B (10% water, 0.1% TFA and 90% ACN) buffers, which achieved 30% ACN for two column volumes and prepared for elution by increasing the mixed concentration of ACN from 30% to 35% over one column volume. Tirezepatide was eluted from the column using an increase of 1.5% ACN per column volume until elution was complete. The eluate was fractionated and the purity was determined using RP-HPLC. The column was regenerated by increasing ACN from 47% to 65% over one column volume and continuously flowing 65% ACN over three column volumes. Before the next injection sequence, the column was re-equilibrated with 30% ACN for two column volumes.

[0107] Pool the fractions that meet the mainstream inclusion conditions. For fractions that do not meet the purity standards but have a purity greater than 50%, they can be combined for loop injection after all single injections are completed. The loop fractions are divided into frontside fractions and backside fractions, which are diluted with buffer A and refrigerated. Use single injection standard processing and pooling loop injection; however, only the main peak fraction is continued to be processed and no further circulation is performed. After completing the mainstream pooling, the concentration and purity of the intermediate are determined. Before RP2 processing, the material is diluted and the pH is adjusted to pH 8. The RP1 method produces 37 kg of crude product and 14277 g of contained product with an average overall purity (pool purity) of 90.9%.

[0108] Reverse phase purification 2: Example 4A RP1 solution was loaded onto a pre-equilibrated Kromasil 100-10-C8 packed column. The column was washed with a mixture of buffer C (90% NH4OAC aqueous solution pH 8.0, 10% ACN) and buffer D (10% NH4OAC aqueous solution pH 8.0, 90% ACN), which resulted in a 20% ACN solution for two column volumes. Tirzepatide was eluted from the column using 3.5% ACN per column until elution was complete. The eluate was fractionated and the purity was determined using RP-HPLC. After elution, the column was regenerated by increasing the ACN to 80% over one column volume and continuing to flow 80% ACN for three column volumes. Before the next injection sequence, the column was re-equilibrated using 20% ​​ACN for two column volumes.

[0109] Pool the fractions that meet the mainstream inclusion criteria. Fractions that do not meet the purity criteria but are greater than 60% pure can be combined for loop injection after all single injections are completed. The recovered fractions are divided into a front pool and a back pool, diluted with buffer C and refrigerated. The loop injection is processed and pooled using the single injection standard; however, only the main peak fraction will be processed further and no further loops are performed. After the mainstream pooling is completed, the concentration and purity of the intermediate are determined. The pH of the material can be adjusted to 8.0 in preparation for the TFF step. The RP2 method started with 14.2 kg and obtained 10.9 kg of included product with a yield of 76.7%.

[0110] Ion Exchange Chromatography (IEX): The Example 4A RP2 solution was filtered and loaded onto an Amberchrom CG300M column. Two fractions were eluted using mobile phase E (10% ammonium acetate in water, 5% IPA, pH 8) and mobile phase F (isopropanol). Fractions were analyzed for peptide content, and those with <3 mg / mL were discarded. The concentrated pooled fractions were stored at 20°C prior to precipitation. The IEX process, starting with 10.9 kg of RP2, provided 14.3 kg of Example 4A material with an overall purity of 97.8%.

[0111] Precipitation: The Example 4A IEX solution (333 kg) was filtered and isopropanol (850 L) was added to reduce the water content to <10% w / w water. The diluted solution was cooled to 0±3°C in preparation for the addition of MTBE and precipitation. MTBE (2304 L, 1708 kg) was cooled to 0±3°C. During the first ~37% of the MTBE charge, cold MTBE was fed to the IEX solution at a rate of ~0.69 kg / min. The feed rate was then increased to an average of ~2.3 kg / min to complete the remaining ~63% of the MTBE charge. The temperature during the feeds was maintained at <5°C. The resulting precipitate slurry was cold filtered (≤-10°C). The filter cake was then washed with MTBE. The filter cake was dried to LOD <2%.

[0112] Humidification of 4A: Example 4A was humidified by passing wet nitrogen through the filter dryer. The humidity of the filter outlet stream was monitored every 60 minutes. Humidification was continued until <0.5% MTBE and <0.2% IPA remained in the wet cake. After the humidification process was complete, the nitrogen flow was switched to allow dry nitrogen to flow through the pure Example 4A product cake. The material was sampled for water and residual solvent according to the specific IPC target and continued to dry with dry nitrogen until the desired target water content of 5-7% w / w was achieved. A total of 12.9 kg of Example 4A was isolated with a peptide content purity of >95%. The total yield based on Sieber resin loading = 31%.

[0113] Example 4B (linear SPPS) Tirzepatide (SEQ ID NO: 1) Preparation 23 The method for producing Preparation 23 was essentially as described in Example 4A, except that NMP was replaced entirely with DMF in all couplings and deprotections. In addition, the stoichiometric ratio of amino acid:Oxyma to DIC was reduced to 2.5:2.5:2.7 molar equivalents based on Siber resin. The only exception related to the use of DMF was the coupling of Ile 12 with Aib 13, in which NMP was retained. In this example, 17.6 kg of Sieber resin was coupled to 92.2 kg of the Preparation 23 peptide intermediate on resin during the method.

[0114] Preparation 24 92.1 kg of Preparation 23 was prepared essentially as described in Example 4A; further processing gave 97.3 kg of the peptide intermediate on resin, Preparation 24. Preparation 24 was packaged and refrigerated (-20°C) prior to cleavage from the resin.

[0115] Resin cleavage and crude isolation of Example 4B: Two batches were run on a 32 kg scale of Preparation 24 using essentially the same conditions as described for Example 4A to provide 24.4 kg of Example 4B with 69.5% HPLC purity and 52.6% yield; and 21.3 kg of Example 4B with 88.3% HPLC purity and 45.2% yield. The crude intermediate was refrigerated (-20° C.) until purification.

[0116] Example 4B Purification: Crude Dissolution: Crude Tirzepatide Example 4B was charged to a dissolution vessel and dissolved in a 1:1 acetonitrile:water solution to a final concentration of 25 g solids / L of solution. The pH of the resulting solution was adjusted to 8.5-9.5 with ammonium hydroxide to initiate the conversion of the depsipeptide isomer (10-15%) to Tirzepatide Example 4B. The pH-adjusted mixture was stirred for at least one hour to allow the depsipeptide conversion to occur. The pH was then adjusted to 1.5-2.5 by adding trifluoroacetic acid and diluted to 30% acetonitrile content in preparation for chromatography. In total, the crude solution was stirred for at least 7 hours to convert the Trp CO2 salt to Tirzepatide Example 4B.

[0117] Conversion of tirzepatide (TZP) to depsipeptide: Conversion of depsipeptides to APIs: Reverse Phase Purification 1 (RP1): The RP1 method, essentially as described in Example 4A, can be used to convert the depsipeptide into the API. The RP1 purification method is essentially the same as described in Example 4A; however, the crude solubilization step described above enhances the capacity of the RP1 chromatography step. This allows for a higher g of Tirzepatide per L of resin load and reduces the number of injections required to purify crude Tirzepatide under the conditions described in Example 4. In this example, 23.7 kg of content-corrected crude Example 4B yielded 25.4 kg of Example 4B (107%) after RP1. Total solution volume = 2910 L @ 8.72 g / L, and once all pooled fractions were collected, the mixture was stirred, sampled, and allowed to settle before proceeding to Reverse Phase Purification 2 (RP2).

[0118] Reverse Phase Purification 2 (RP2): Reverse Phase Purification 2 (RP2) used essentially the same purification method as described in Example 4A, using methods known to those skilled in the art. In this example, 15.2 kg of Example 4B from RP1 was purified to 13.8 kg of Example 4B after RP2, with a purity of ~98%. Total solution volume = 808 L @ 17.0 g / L. The mixture was stored prior to tangential flow filtration.

[0119] Tangential Flow Filtration (TFF): The TFF membrane was installed and rinsed with water. Ammonium acetate buffer was prepared using low endotoxin purified water, acetic acid, and ammonium hydroxide. Isopropyl alcohol was then added to provide a 5:95 100 mM NH4OAc pH 8.0:IPA buffer. The 17 g / L RP2 solution of Example 4B was concentrated to ~125 g / L by TFF. The RP2 solution was recirculated, allowing the solvent to permeate through the membrane while retaining the peptide solution on the retentate side of the membrane in solution. After concentration, the diafiltration buffer was fed into the retentate storage tank while the permeate was continuously collected. The buffer exchange continued until the desired solvent composition and peptide concentration were achieved. The solution was emptied from the system and the resulting polarization layer was rinsed from the membrane and pooled with the peptide concentrate. Two portions of RP2 solution (403.9 L @ 17 g / L, 9.87 kg API) were processed by TFF.

[0120] Co-feed precipitation: The TFF portions were combined (138.2 kg, 78.3 g / L) and KF (8.9%) was measured to verify <10% water. MTBE (243 kg) was added to a separate container and cooled to 0°C. IPA (48 kg), water (6 kg) and MTBE (100 kg) were added to the precipitation vessel, and the solution was cooled to 0°C. The TFF and MTBE process streams were co-fed to the precipitation vessel at rates of 1.6-1.8 kg / min and 2.9-3.1 kg / min, respectively. The resulting slurry was aged at 0°C for another 0.7 hour and then warmed to 15°C. The slurry was aged at 15°C for 1 hour and then MTBE (118 kg) was added. The slurry was aged at 15°C for 1 hour and then cooled to 2.5°C. The slurry was cold filtered and the filter cake was washed with MTBE (573 kg). The filter cake was dried to LOD <2%.

[0121] Wet-Up: Wet-up was performed essentially as described in Example 4A and applied to the Example 4B material using methods known to those skilled in the art. A total of 14.5 kg of Example 4B (SEQ ID NO: 1) was isolated with an HPLC purity of >97.7% and a peptide content of 88.4%. Overall yield based on Sieber resin loading = 46%.

[0122] Example 5 Continuous synthesis of preparations using convergent chemistry in flow 31 The synthesis of prepared product 31 by peptide fragments uses both chemical batch method and the method of sequentially adding fragments in tubular flow reactor to carry out.The general method of synthesis relates to coupling two fragments by mixing two solutions together with coupling agent in tubular reactor, then is followed by adding alkali, and the extra residence time in tubular reactor to carry out the removal of FMOC protecting group.This is ready for coupling material to add next fragment.Use nanofilter with membrane, remove excessive reagent, alkali and solvent between continuous coupling reactions, described membrane is adjusted in size so that retain peptide and permeate lower molecular weight impurity.Diafiltration is used for removing lower molecular weight impurity fully before subsequent coupling step.Below include the description and analytical results of the example deriving from these conversions.

[0123] HPLC was used to confirm the synthesis of Preparation 31 from Preparation 29 and Preparation 30. The analytical method used a C4 stationary phase column (2.1 mm ID x 150 mm x 1.7 micron particle size) at 65°C with a 60-98% B gradient of 0.1% TFA in water and acetonitrile over 12 minutes. UV detection at 214 nm was used for this material.

[0124] Table A.5 shows high-resolution mass spectrometric data collected on the product of the Step 3 coupling reaction (Preparation 31) performed in flow. The mass accuracy confirmed the desired material.

[0125] Table A.5 Mass accuracy confirmed for measurements of Preparation 31 using calculated high resolution mass spectrometry data.

[0126] 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 segments to produce a transient thioester-linked intermediate. The thioester-linked intermediate rearranges to provide the 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 for the chemical synthesis of full-length peptides containing cysteine ​​or alanine.

[0127] Example 6 Native chemical ligation method Tirzepatide (SEQ ID NO: 1) Synthesis of Fmoc-hydrazine-CTC resin (Preparation 32) 2-CTC resin (10.7 g, 17.7 mmol) was swelled in 100 mL of DCM at 0°C for 20 min. 9-Fluorenylmethyl carbazate (15.6 g, 61.4 mmol, 3.5 eq) was dissolved in 210 mL of 2:1 DMF:DCM. DIEA (31 mL, 178 mmol, 10.1 eq) was added to the 9-Fluorenylmethyl carbazate solution. The solution was then slowly added to the resin at 0°C. The mixture was stirred at 0°C for about one hour and then warmed to room temperature. The reaction mixture was stirred at room temperature for 16 hours. 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 hours. The resin loading was determined to be 0.74 mmol / g by quantitative NMR.

[0128] Synthesis of peptide hydrazide (17-mer) (Preparation 33) SEQ ID NO: 32 Hydrazine-CTC resin (1.01 g, loading value: 0.65 mmol / g) was placed in a 40 mL reactor vessel and swollen with 3×4 mL DCM (30 s each), then swollen with 2×10 mL 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 DMF. DIPEA (0.91 mL, 5.22 mmol, 8 equiv) 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 5×10 mL DMF, 5×10 mL DCM, and dried for 12 hours. The loading of the resulting resin was 0.54 mmol / g as measured by quantitative NMR. 0.91 g of this resin was used for the synthesis of Preparation 33 (SEQ ID NO: 32).

[0129] Deprotection: 4 x 9 mL of 20% v / v piperidine in DMF, 30 min each.

[0130] Coupling: For amino acid coupling, use 3 equivalents of amino acid, 3 equivalents of OXYMA, and 3.3 equivalents of DIC. After each coupling and the final deprotection, wash the resin with 5 × 9 mL of DMF and 1 min of N₂. At the end of the peptide hydrazide synthesis, wash the resin with DCM under N₂. Dry the resin on the synthesizer.

[0131] Deprotection and cleavage: 25 mL of a cleavage mixture 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 precipitated immediately. The filter flask was washed with 2×2.0 mL TFA and poured into cold MTBE. It was cooled to -20°C and held for half an hour before centrifugation. 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 preparation 33 was obtained [expected (mass + 2H + ) / 2=968.4883, observed (mass + 2H + ) / 2=968.4879].

[0132] About 0.62 mmol of Preparation 34 was synthesized on Sieber amide resin by standard SPPS protocol. Fmoc-Lys(ivDde)-OH was used for orthogonal deprotection and lysine acylation.

[0133] Deprotection of ivDde: Dilute hydrazine monohydrate (64% w / w) (1.98 g, 25.3 mmol) to 24.4 g with DMF and add 20 g to the resin. Stir the slurry with a stream of nitrogen. After approximately 2 hours, wash with 5 x 9 mL of DMF. Repeat once more.

[0134] 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 Preparation 6 was then added to the resin and mixed with a stream of nitrogen for 12 hours. After 12 h, the solution was drained and the resin was washed with 5 x 10 mL DMF and 7 x 10 mL DCM mixed with 1 min N 2 . The resin was dried on the synthesizer for 8 h.

[0135] 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 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 of TFA. The filtrate was poured into 200 mL of cold MTBE, which immediately precipitated the peptide. The filter flask was washed with 2 x 2 mL of TFA and poured into cold MTBE. This 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 Preparation 34 was obtained. This was purified by RP-HPLC on a Kromasil 100-10-C8 10 μm column (30 mm x 250 mm) at ambient temperature. The purification was a linear gradient of 15% acetonitrile in water for the first 5 minutes, 30-55% acetonitrile in water over the next 25 minutes, and a constant 0.1% TFA over 30 minutes. 1.28 g of purified Preparation 34 (SEQ ID NO: 33) was obtained [expected (mass + 2H + ) / 2=1470.7929, observed (mass + 2H + ) / 2=1470.7885].

[0136] Thioester synthesis (conversion of Preparation 33 to Preparation 35) The crude peptide hydrazide (Preparation 33, 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 allowed to stir 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 allow in situ thiolysis of the peptidyl azide generated by Preparation 33.

[0137] The pH of the reaction mixture was adjusted to approximately 6.95 with 5N sodium hydroxide solution. 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 at ambient temperature on a Waters X-Bridge C18 10 μm column (10 mm × 250 mm) with a linear gradient of 10% acetonitrile in water for the first 2.8 min, 25-42% acetonitrile in water for the next 25 min, and a constant 0.1% TFA over 28 min. This gave 1.03 g of TFET thioester (Preparation 35 (SEQ ID NO: 34)) [expected (mass + 2H + ) / 2=1010.4650, observed (mass+2H + ) / 2=1010.4620].

[0138] Native chemical ligation: An aqueous solution of 6 M guanidine hydrochloride and 0.3 M sodium dihydrogen phosphate (pH 7.0) was the ligation buffer used in native chemical ligation. All solutions were prepared in this ligation buffer. 350.4 mg (0.174 mmol) of peptide thioester Preparation 35 (SEQ ID NO: 34) was dissolved in 50 mL of ligation buffer. An 8.0 mL portion of 0.5 M 4-mercaptophenylacetic acid (MPAA) solution was added to the peptide thioester solution. A peptide containing an N-terminal cysteine ​​(Preparation 34 (SEQ ID NO: 33), 524.6 mg, 0.178 mmol, 1.03 equiv) was dissolved in 48 mL of ligation buffer in a 50 mL centrifuge tube. The solution of Preparation 34 was added to the thioester solution. The 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. An 8.0 mL portion 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. Before purification, an additional 3 mL of 0.5 M TCEP solution was added. Preparation 36 (SEQ ID NO: 35) was purified by RP-HPLC on a Kromasil 100-10-C8 10 μm column (10 mm x 250 mm) at ambient temperature using a linear gradient of 10% acetonitrile in water for the first 4 minutes followed by 20-50% acetonitrile in water (0.1% acetic acid, titrated to pH 9.0) over a 28 minute purification period. Approximately 372 mg (44.3%) of the tirzepatide cysteine ​​analog Preparation 36 [expected (mass + 3H+ ) / 3=1615.17263, observed (mass + 3H + ) / 3=1615.1686].

[0139] Desulfurization: The buffer used in the desulfurization was an aqueous solution of 6-guanidine hydrochloride and 0.3 M sodium dihydrogen phosphate (pH 7.0). All solutions were prepared in this buffer. 2,2'-Azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (Preparation 37, 808.2 mg, 2.5 mmol) was dissolved in 10 mL of buffer and the pH was adjusted to approximately 7.0 with 5N NaOH. The volume was made up to 15 mL with buffer. Tirzepatide cysteine ​​analog Preparation 36 (105.2 mg, 0.022 mmol) was dissolved in 30 mL of buffer, and 6 mL of Preparation 37 solution was added. Five mL of 0.3 M L-glutathione reduced 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 hours, whereupon UPLC analysis revealed that the reaction was complete [expected (mass + 3H + ) / 3=1604.5153, observed (mass + 3H + ) / 3=1604.5122]. The desulfurization yield was calculated by UPLC using tirzepatide (SEQ ID NO: 1) as a reference standard. The estimated yield was 47%.

[0140] Native chemical ligation (method 2): synthesis of peptide hydrazide preparation 39 SEQ ID NO: 36 Hydrazine-CTC resin (2.03 g, 1.32 mmol, loading: 0.65 mmol / g) was placed in a 40 mL reactor vessel and swollen on a Symphony synthesizer with 3 x 10 mL DCM (30 s each) followed by 2 x 10 mL DMF (20 min each). 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-tetraazatricarboxane-53-oic acid (Preparation 17, 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 of Preparation 17 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 DMF, 5×10 mL DCM, and dried for 12 hours. The loading of the resulting resin was measured to be 0.26 mmol / g by quantitative NMR. 1.82 g of this resin was used for the synthesis of peptide hydrazide preparation 39 (SEQ ID NO: 36).

[0141] Deprotection: 4 x 9 mL 20% v / v piperidine in DMF, 30 min each.

[0142] Coupling: 3 equivalents of amino acid, 3 equivalents of OXYMA and 3.3 equivalents of DIC were used for amino acid coupling.

[0143] After each coupling and the final deprotection, the resin was washed with 5 × 9 mL of DMF mixed with 1 min of N2. At the end of the peptide hydrazide synthesis, the resin was washed with 7 × 10 mL of DCM mixed with 1 min of N2. The resin was then dried on the synthesizer for approximately 12 h.

[0144] 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 poured into 175 mL of cold MTBE. The filter flask was washed with TFA (2 x 2.5 mL), and the washes poured into cold MTBE. This was cooled to -20°C for 30 minutes and 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 preparation 39 (SEQ ID NO: 36) was obtained. The crude peptide hydrazide, Preparation 39, was purified by RP-HPLC on a Waters XSelect CSH C18 10 μm column (10 mm x 250 mm) at ambient temperature with a linear gradient of 10% acetonitrile in water for the first 3 minutes, 20-55% acetonitrile in water over 23 minutes, and a constant 0.1% TFA over 28 minutes. Approximately 110 mg of partially purified hydrazide, Preparation 39, was obtained.

[0145] Deprotection and Cleavage: 25 mL of a cleavage cocktail 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 of TFA. The filtrate was poured into 200 mL of cold MTBE, and the peptide immediately precipitated. The filtrate flask was then washed with 2 x 2 mL of TFA, and the washes were poured into cold MTBE. This was cooled to -20°C for 30 minutes and then 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 preparation 40 (SEQ ID NO: 37) was obtained.

[0146] Native chemical ligation: An aqueous solution of 6M guanidine hydrochloride and 0.3M sodium dihydrogen phosphate (pH 7.0) was the ligation buffer used in native chemical ligation. All solutions were prepared in this ligation buffer. Partially purified peptide hydrazide (preparation 39, 56 mg, 0.019 mmol) was dissolved in 5 mL of ligation 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 1 M sodium nitrite solution (0.25 mmol, 13.2 equivalents) 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 cause in situ thiolysis of the peptidyl azide produced by preparation 39. The pH of the reaction mixture was adjusted to approximately 7.0 with 5N sodium hydroxide solution. The thiolysis of the peptidyl azide was allowed to proceed for 30 min.

[0147] About 0.62 mmol of preparation 40 (SEQ ID NO: 37) was synthesized on Sieber amide resin using a standard SPPS protocol. Preparation 40 (26.1 mg, 0.014 mmol, 0.74 quiv) containing an N-terminal cysteine ​​was dissolved in 1 mL of ligation buffer. The solution of preparation 40 was added to the thioester solution. The vial containing preparation 40 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 5N sodium hydroxide solution. The reaction was stirred at room temperature for one hour. Tirzepatide cysteine ​​analog preparation 42 was observed in the reaction mixture.

[0148] Sequence SEQ ID NO:1 Tirzepatide wherein X1 is Aib; X2 is Aib; K at position 20 is replaced by (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γGlu)1-CO-(CH2) 18 -CO2H was conjugated to the ε-amino group of the K side chain and chemically modified; and the C-terminal amino acid was amidated to form a C-terminal primary amide. SEQ ID NO:2 SEQ ID NO:3 SEQ ID NO:4 SEQ ID NO:5

[0149] SEQ ID NO:6 SEQ ID NO:7 SEQ ID NO:8 SEQ ID NO:9 SEQ ID NO:10 SEQ ID NO:11 SEQ ID NO:12 SEQ ID NO:13 SEQ ID NO:14 SEQ ID NO:15 SEQ ID NO:16 SEQ ID NO:17 SEQ ID NO:18 SEQ ID NO:19 SEQ ID NO:20 SEQ ID NO:21 SEQ ID NO:22 SEQ ID NO:23 SEQ ID NO: 24 SEQ ID NO:25 SEQ ID NO:26 SEQ ID NO:27 SEQ ID NO:28 SEQ ID NO: 29 SEQ ID NO:30 SEQ ID NO: 31 SEQ ID NO: 32 SEQ ID NO: 33 SEQ ID NO:34 SEQ ID NO: 35 SEQ ID NO:36 SEQ ID NO:37 HCFVQWLIAGGPSSGAPPPS-NH2 SEQ ID NO: 38 SEQ ID NO: 39 SEQ ID NO: 40 Specifically, the present application relates to the following items: Item 1: a compound of SEQ ID NO: 17 or a pharmaceutically acceptable salt thereof. Item 2: The compound of SEQ ID NO: 11 or a pharmaceutically acceptable salt thereof. Item 3: The compound of SEQ ID NO: 22 or a pharmaceutically acceptable salt thereof. Item 4: The compound of SEQ ID NO: 21 or a pharmaceutically acceptable salt thereof. Item 5: The compound of SEQ ID NO: 20 or a pharmaceutically acceptable salt thereof. Item 6: Compounds of the following formula: or a pharmaceutically acceptable salt thereof. Item 7: The compound of SEQ ID NO: 2 or a pharmaceutically acceptable salt thereof. Item 8: Compounds of the following formula: or a pharmaceutically acceptable salt thereof. Item 9: The compound of SEQ ID NO: 4 or a pharmaceutically acceptable salt thereof. Item 10: The compound of SEQ ID NO: 7 or a pharmaceutically acceptable salt thereof. Item 11: The compound of SEQ ID NO: 14 or a pharmaceutically acceptable salt thereof. Item 12: The compound of SEQ ID NO: 17 or a pharmaceutically acceptable salt thereof. Item 13: The compound of SEQ ID NO: 33 or a pharmaceutically acceptable salt thereof. Item 14: The compound of SEQ ID NO: 32 or a pharmaceutically acceptable salt thereof. Item 15: The compound of SEQ ID NO: 34 or a pharmaceutically acceptable salt thereof. Item 16: The compound of SEQ ID NO: 35 or a pharmaceutically acceptable salt thereof. Item 17: The compound of SEQ ID NO: 36 or a pharmaceutically acceptable salt thereof. Item 18: The compound of SEQ ID NO: 38 or a pharmaceutically acceptable salt thereof. Item 19: The compound of SEQ ID NO: 39 or a pharmaceutically acceptable salt thereof. Item 20: A method for preparing tirzepatide or a pharmaceutically acceptable salt thereof, comprising nanofiltration of the intermediate tirzepatide. Item 21: The method of Item 20, wherein the intermediate tirzepatide is SEQ ID NO: 27. Item 22: The method of Item 20, wherein the intermediate tirzepatide is SEQ ID NO: 29. Item 23: The method of any one of items 20 to 22, wherein the nanofiltration method comprises diafiltration. Item 24: The method of any one of items 20 to 23, wherein the method comprises DMF diafiltration. Item 25: A method for preparing tirzepatide or a pharmaceutically acceptable salt thereof, comprising deprotecting the compound of SEQ ID NO: 22 or a pharmaceutically acceptable salt thereof. Item 26: The method described in Item 25, wherein the deprotection solution contains dithiothreitol, triisopropylsilane and trifluoroacetic acid. Item 27: A method for selectively acylating lysine amino acids in peptides, comprising coupling resin-bound peptide-lysine-NH2 with tert-butyl-eicosanedioyl-Glu-(O-tert-butyl)-(8-amino-3,6-dioxaoctanoic acid)-(8-amino-3,6-dioxaoctanoic acid)-OH. Item 28: The method of Item 27, wherein the peptide is an incretin. Item 29: The method of any one of Items 27 or 28, wherein the resin-bound peptide-lysine-NH2 is SEQ ID NO: 24 or a pharmaceutically acceptable salt thereof. Item 30: A method for converting a depsipeptide isomer into a desired peptide, comprising: a. adjusting the pH of the depsipeptide isomer to about pH 7 to about pH 10; and b. Incubate the depsipeptide isomers at pH 7 to pH 10 for at least one hour. Item 31: The method of Item 30, wherein the depsipeptide isomer is adjusted to about pH 8.5 to about pH 9.5. Item 32: The method of any one of Items 30 and 31, wherein the peptide is an incretin. Item 33: The method of any one of items 30 to 32, wherein the depsipeptide isomer is recovered from a process waste stream. Item 34: The method of any one of items 30 to 33, wherein the depsipeptide isomer is SEQ ID NO: 40 or a pharmaceutically acceptable salt thereof. Item 35: A method for desulfating incretin or a pharmaceutically acceptable salt thereof, comprising contacting the incretin with a free radical initiator. Item 36: The method described in Item 35, wherein the free radical initiator is a water-soluble free radical initiator. Item 37: The method of any one of Items 35 or 36, wherein the free radical initiator is an azo initiator. Item 38: The method of any one of Items 35 to 37, wherein the free radical initiator is selected from 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride and 2,2'-azobis(2-methylpropionamidine) dihydrochloride. Item 39: The method of any one of Items 35 to 38, wherein the incretin is SEQ ID NO: 35 or a pharmaceutically acceptable salt thereof. Item 40: The method of any one of items 35 to 39, wherein the desulfurization method provides the peptide of SEQ ID NO: 1.

Claims

1. A method for preparing tirzepatide or a pharmaceutically acceptable salt thereof, comprising deprotecting the compound of SEQ ID NO: 22 or a pharmaceutically acceptable salt thereof.

2. A method for selectively acylating lysine amino acids in a peptide, comprising coupling a resin-bound peptide-lysine-NH2 with tert-butyl-eicosanedioyl-Glu-(O-tert-butyl)-(8-amino-3,6-dioxaoctanoic acid)-(8-amino-3,6-dioxaoctanoic acid)-OH.

3. A method as claimed in claim 2, wherein the peptide is an incretin.

4. A method for converting a depsipeptide isomer into a desired peptide, comprising: a. adjusting the pH of the depsipeptide isomer to about pH 7 to about pH 10; and b. Incubate the depsipeptide isomers at pH 7 to pH 10 for at least one hour.

5. A method for desulfating an incretin or a pharmaceutically acceptable salt thereof, comprising contacting the incretin with a free radical initiator.

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