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 an invention patent application with an application date of January 28, 2020, application number 202080011536.7, and name “Method for Preparing GIP / GLP Dual Agonists”.

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

[0002] Diabetes is a chronic disease characterized by hyperglycemia 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 glucose levels. Tirzepatide, a dual GIP / GLP1 agonist, is described and claimed in U.S. Patent 9,474,780 ("the '780 patent"). Tirzepatide can be used to treat T2D.

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

[0004] Methods and intermediates are needed to enable the technology of producing tirzepatide to be improved, so that the technology has multiple advantages including commercially required purity. Similarly, efficient and environmentally friendly "green" methods are also needed, including stable intermediates, so as to provide tirzepatide with fewer purification steps. For the purpose of improving the safety of the environment and operators, improved technology is also needed to provide a method for preparing tirzepatide that produces minimal waste streams. The preparation of large-scale, pharmaceutically elegant tirzepatide has many technical challenges that may affect total yield and purity. It is necessary to avoid the use of transition metals and / or methods for harsh reaction conditions that are incompatible with peptide synthesis.

[0005] The present invention seeks to meet these needs by providing novel intermediates and methods that can be used to prepare tirzepatide (SEQ ID NO: 1) or a pharmaceutically acceptable salt thereof. The improved tirzepatide preparation methods 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 a 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 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 following 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 of SEQ ID NO: 40 or a pharmaceutically acceptable salt thereof.

[0018] The present invention provides free radical based desulfurization, which comprises 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 isopropanol, “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 hexafluoroisopropylsilane. 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, and "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-diimide)-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 the font is generally smaller 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 a sequence.As used herein, the term "protected" means that a protecting group is attached at the indicated position. One skilled in the art will recognize that a variety of protecting groups are well known and that alternative protecting groups may be suitable for a particular process.

[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 resins well known to those skilled in the art for preparing the peptides disclosed herein; however, alternative resins may be selected for 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 fluorenylmethoxycarbonyl chloride (Fmoc) peptide chemistry techniques using sequential couplings with an automated peptide synthesizer. The resin was swollen with DMF and then deprotected with 20% piperidine (Pip) / DMF (3×30 min). Subsequent Fmoc deprotection used 20% Pip / DMF 3×30 min treatments, and 4×30 min treatments were used for more difficult couplings. After deprotection, the resin was washed with 5×2 min, 10 volumes of DMF washes. Amino acid preactivation used diisopropylcarbodiimide (DIC) / ethyl cyanohydroxyiminoacetate (Oxyma) DMF solution 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 with 5×2 min 10 volumes of DMF. To isolate the final product, the resin-bound product was washed with 10 volumes of DCM for 5×2 min to remove DMF. The resin was washed with 2×2min 10 volumes of IPA to remove DCM, washed with 5×2min 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, and the resin was then 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 min, then 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 in vacuo 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]

[0136] Preparation 1 Soft Cleavage: 10 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 sintered 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, and 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) Combine the filtrates and transfer to a round bottom flask containing 1000 mL of MTBE. 9) Concentrate the resulting suspension to a light yellow oil, triturate with 200 mL of MTBE, and cool in an ice bath for 30 minutes. 10) Filter the solid, wash with 50 mL of cold MTBE, and dry in a vacuum oven at 33° C. overnight to produce 5.35 g (91% yield) of a white solid. UPLC was used to analyze the isolated solid (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 was added resin-bound Preparation 2 (3.06 g, 1.12 mmol) and 30 mL of 30% HFIP DCM solution, where 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: Add resin-bound preparation 4 (2.0 g, 0.62 mmol) and 10 mL of 30% HFIP DCM solution to a 20 mL glass scintillation vial, where a red color change is observed. The vial is agitated by rotating on a wheel at ambient temperature, then the resin is filtered out, washed with 3×2 mL DCM, and the solvent is removed in vacuo to form a glassy, ​​viscous foam. The foam is dissolved in 5.2 mL DMSO. The solution with 6 mL of water is added over 45 min with 1 mL of water at an equal flow rate (T~15° C.). Once the peptide solution is fully added, an additional 6 mL of water is added over 45 min. A white solid precipitates after addition. The resulting slurry is stirred at 15° C. for 30 min. The solid is filtered, washed with 6 mL of water, and then transferred to a vacuum oven at 35° C. and 25 torr. This produces preparation 4 (Boc-1-14-OH, 1.0763 g), which is 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 at the same time. 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, 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 then washed with water (2×4 mL). The wet cake was dried at 35° C. under vacuum 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, the measured value is 5608.2066).

[0031] Synthesis of Preparation 6 using Method 1 (LPPS) Docosane diacid 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 byproduct 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, and then a solution of TMSA (11.3 kg) dissolved in DCM (7 L) was added, and the reaction mixture was stirred at 40 ° C for 1 hour. INT1 (17.0 kg) DCM solution was added at room temperature and stirred for 8 hours. After the reaction was completed, 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] Dissolve INT 2 (17.3 kg) and N-hydroxy-succinimide (4.1 kg, 1.2 eq) in ethyl acetate (336 kg) at 27 ° C. Add a solution of DCC (8.33 kg, 1.25 eq) in ethyl acetate, and stir the reaction at 22 ° C for 24 hours. Filter out the resulting DCU by-product. Concentrate the organic phase, co-evaporate with isopropanol, and then crystallize by cooling the isopropanol solution (~125 L). Thereafter, rinse the filter cake with cold isopropanol and dry at 25 ° C to provide 16.3 kg INT 3, which is 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 completed, the mixture was concentrated and then exchanged with ethyl acetate. The organic phase was washed three times with 2% KHSO4 / NaCl aqueous solution (~200 L), and then washed 4 times with 2% NaCl aqueous solution (~200 L) to target pH 4.5. The organic phase was concentrated and exchanged with acetonitrile. The acetonitrile solution was cooled to -20 ° C, and the resulting suspension was aged at -20 ° C for 15 hours. The mixture was filtered and the filter cake was rinsed with cold acetonitrile and then dried at <0°C to provide 18.4 kg of Preparation 6 (88% yield) with 96% purity. Overall yield = 53%.

[0035] Synthesis of Preparation 6 by 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 DMF swelling followed by Pip / DMF; DMF wash; and amino acid, DIEA, HATU mixture; and DMF wash cycle followed by drying.

[0040] The resin is cracked by stirring the combined batch in 30% HFIP / DCM (240mL) for 1.5 hours. The resin is filtered, washed, and the solvent is removed from the filtrate in a vacuum. The gained oil is dissolved in acetonitrile and the solvent is removed again. This operation provides 30.47g (146% of theoretical yield) of viscous yellow oil, which contains 52.3 area % desired product analyzed by UPLC. The crude product is purified by flash chromatography (500 grams of silica gel, eluted with 85% DCM / 10% methanol / 5% acetic acid, 38 × 100mL fractions are collected). The previous chromatographic concentrate (17.94g) is crystallized to produce 13.4g (74.7% yield), and the UPLC purity it has is 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 equiv). 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 and the resulting solid was added with 50 μL of a solution of trifluoroacetic acid (4.65 mL), triisopropylsilane (20 μL) and DTT (20 mg). 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, 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) for about 20min and then drained. Wash the obtained resin with DMF (80mL, 6 volumes) for about 5min. Remove the Fmoc group by treating Fmoc-amino acid resin twice (10min and 20min respectively) with 5 volume % piperidine, 1.25 volume % DBU, 1.0wt.% HOBt / DMF solution (80mL, 6 volumes). After draining the de-Fmoc solution, wash with DMF (80mL, 6 volumes) twice, MTBE (80mL, 6 volumes) twice, and then with DMF (80mL, 6 volumes) twice.

[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 wet, 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). Allowed to stand at 0 °C-5 °C for about 5 minutes. DCM (60 mL, 1.5 volumes) was added to the resin, followed by the addition of an activated Fmoc-amino acid solution. The resulting mixture was stirred at about ambient temperature for 2 hours. The Fmoc removal process was repeated and coupled with the remaining amino acids in order. After the last Fmoc removal process 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 give the preparation 9-Seiber (21.21 g, 0.435 mmol / g theory, 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 vol% TFA (101 mL, 10 vol) in DCM and a DCM wash step. The cleavage fractions and washes 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 to give a white slurry product. 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) for 20min, then drained. Wash the obtained resin with DMF (108mL, 6 volumes) for 5min. Remove the Fmoc group by processing Fmoc-amino acid resin twice (respectively 10min and 20min) with 5 volume % piperidines, 1.25 volume % DBU, 1.0wt.% HOBt / DMF solution (108mL, 6 volumes). Drain off-Fmoc solution, and use DMF (110mL, 6 volumes) twice, MTBE (110mL, 6 volumes) twice and then use DMF (110mL, 6 volumes) twice washing resin. 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) twice for 5 min, 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 a 1 vol% solution of TFA in DCM (100 mL, 10 vols) and washed with DCM (75 mL, 7.5 vols). 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% volume of the combined filtrate) followed by concentration to dryness. Finally, the peptide was triturated with stirring in deionized water (150 mL, 40% 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), separated, 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, 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 about 20 minutes, then drained. The obtained resin was washed with DMF (400 mL, 10 volumes) for 5 min twice. The amino acid chain was assembled using standard Fmoc chemistry. 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 obtained solution was cooled to about <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 about 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] The Fmoc group was removed by treating the Fmoc-amino acid resin twice (10 min and 20 min each) with 5 vol% piperidine, 1.25 vol% DBU, 1.0 wt% HOBt / DMF solution (240 mL, 6 volumes). The de-Fmoc solution was drained, and the resin was washed twice with DMF (240 mL, 6 volumes), MTBE (240 mL, 6 volumes), and DMF (240 mL, 6 volumes). The peptide resin was fully washed with 2-propanol (400 mL, 10 volumes) twice and MTBE (400 mL, 10 volumes each, 6 times) after completing the synthesis step, and then dried under vacuum at 35°C to produce a loaded resin (74.82 g, 0.159 mmol / g, 11.90 mmol, 58.9% yield) that was not the final amino acid. The final amino acid Fmoc-Leu-OH was added to a portion of the resin (13.61 g, 0.159 mmol / g, 2.16 mmol) respectively. The resin was swollen with DMF (130 mL, 10 volumes, 3 times) for >5 min each time, then deprotected at 10 min and 20 min (130 mL of the deprotection mixture was prepared from 5.6 g piperidine, 1.67 g DBU, 1.3 g HOBt in 120 mL DMF, 10 volumes twice). The resin was washed with DMF (80 mL, 6 volumes, twice), followed by MTBE (80 mL, 6 volumes, twice), then DMF (80 mL, 6 volumes, twice) for 5 min each. Dissolved in DMF (50 mL, 3.7 vols), followed by 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 adding 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 addition of the activated Fmoc-amino acid solution. The resulting mixture was stirred at about ambient temperature for 2 h. 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 of TFA in DCM (70 mL, 10 volumes) three times for 10 min each at about 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 value 2361.4031, found value 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 (19.16 g, 0.54 mmol / g, 10.35 mmol) was suspended in DMF (190 mL, 10 volumes) for 20 min and then drained. The obtained resin was washed with DMF (190 mL, 10 volumes) for 5 min and then drained. Piperidine (77.82 g), DBU (23.16 g), HOBt (18.09 g, 80 wt%) and DMF (1800 mL) were combined to provide a solution of 5% piperidine, 1.25% DBU, 1.0% HOBt / DMF as a deprotection solution. The Fmoc group was removed by treating the Fmoc-amino acid resin twice (10 min and 20 min each) with a deprotection solution (190 mL, 10 volumes). The de-Fmoc solution was drained and then washed with DMF twice, MTBE twice, and DMF twice (190 mL, 10 volumes each time) to wash the resin.

[0052] DIEA (2.62 g, 20.3 mmol, 2.0 equivalents) was added to a solution of Fmoc-Ile-OH (7.11 g, 10.1 mmol, 2.0 equivalents) in DMF (85 mL). The resulting solution was cooled to 0-5 ° C, 6-chloro-benzotriazole-1-yloxy-tripyrrolidinophosphonium hexafluorophosphate (PyClock) (11.36 g, 20.06 mmol, 2.0 equivalents) was added and dissolved completely. After standing for 3 to 5 minutes, the activated solution was added to the H-Aib-O-CTC resin complex pre-swollen in DCM (30 mL, 1.5 volumes). The reaction was warmed to ambient temperature and stirred for 2 hours. As shown in the assay analysis, the unreacted material was about 18%. Washed with DMF twice, MTBE twice and DMF twice (190 mL, 10 volumes each time). A solution of Fmoc-Ile-OH (10.63 g, 30.08 mmol, 6 eq) in DMF (165 mL) was added to Oxyma (50 mL, 0.6 M in DMF, 30 mmol, 6 eq) and DIC (50 mL, 0.66 M in DMF, 33 mmol, 6.6 eq). Stirred for 5 min at about ambient temperature, 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 hours. The resin was washed with DMF (5 x 140 mL, 7 vols), more DMF (2 x 180 mL, 9 vols), MTBE (2 x 180 mL, 9 vols), then DMF (2 x 180 mL, 9 vols).

[0053] For the 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 ° C with an ice bath, and activate by adding TBTU (6.45g, 2.0 equivalents) and leaving standstill for 3-5min at 0-5 ° C. Add DCM (30mL) to resin, then add activated Fmoc-amino acid solution. Stir the resulting mixture at room temperature for 2 hours. Wash the resulting resin with DMF twice, MTBE twice and DMF twice (190mL, 10 volumes each time) again. Remove the Fmoc group by processing 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] Tetramer Boc-Y-Aib-E(tBu)G-OH (12.25 g, 2.0 eq) was activated with Oxyma (30 mL, 0.6 M in DMF, 20 mmol, 2 eq) and DIC (33 mL, 0.66 M, 22 mmol, 2.1 eq) in DMF (50 mL) for 5 min to add the final four amino acids as a tetramer. The 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 eq) in DMF (40 mL) was added and activated with PyBOP (5.77 g, 1.1 eq) and DIEA (3.32 g, 2.6 eq) 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 (190 mL, 5 min each, 5 times). 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 the chain assembly was completed, 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 three times for 10 min each with 1 vol% TFA in DCM (160 mL, 10 vol) at ambient temperature followed by washing with DCM (120 mL, 7.5 vol). The cleavage fractions and washes 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% volume of the combined filtrate) followed by concentration to dryness. The peptide was mechanically stirred in deionized water (242 mL, 40% volume of the combined filtrate) for 10 min. The solid crude peptide was collected by filtration and washed with deionized water (4×100 mL). The solid was washed with n-heptane (4×100 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, the measured value is 2000.1968).

[0055] Synthesis of Preparation 13 SEQ ID NO: 13 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) were added to a flask under N2 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 about 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 about one hour to provide a conversion to Preparation 13 of about >99%. 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 An aliquot of Preparation 11 (3.012 g, 48.7 wt%, 0.621 mmol, 1.00 equiv) was added to a flask with 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) under N2. The mixture was stirred until a golden solution was produced. Ice water was used 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 about 22 hours to convert to Preparation 14. This resulted in a conversion of about >96%. Piperidine (530 mg, 6.22 mmol, 10.0 equiv) was added to the cooled reaction mixture at a temperature of <10°C. The mixture was stirred at ambient temperature for about 2 hours to provide a conversion of > about 99% to Preparation 14. The reaction mixture was added to another flask containing <4°C 0.5N HCl aqueous solution (12.72 g, 6.23 mmol, 10.0 equiv) and deionized water (16.71 g) to provide precipitation of Preparation 14. The cold slurry was stirred for about 15 minutes, and the white slurry was filtered. The wet cake was 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 about ambient temperature to provide about 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 A sample of TFA (19.656 g, 13.03 mL) was added to a flask with DCM (815 mg, 0.62 mL), DTT (434 mg) and TIPS (362 mg, 0.47 mL) under N2. The mixture was cooled in ice water before adding water (468 mg, 0.47 mL). A sample of Preparation 15 (1016 mg, 39.0 area %, 0.0620 mmol) was added to the mixture at 2°C to provide a solution. The mixture was warmed to about ambient temperature and stirred for about 2 hours. The reaction mixture was added to -15°C MTBE (150 mL) and the reactor was rinsed with MTBE (3 mL). The slurry was centrifuged after about 10 minutes and the supernatant was decanted. The wet cake was re-slurried in MTBE (3×50 mL), centrifuged for each wash, 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 Expected value 4810.5249, measured value 4810.5642), 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 as described herein. The concentrated material was reconstituted with ethanol (5% volume of the combined filtrate) and concentrated to dryness. The peptide was ground 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: A sample of the peptide intermediate on 42.13g of resin was 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 was quenched by adding 4.4mL of pyridine. The resulting solution was combined and concentrated in vacuo. Reconstructed with ethanol (25mL), then concentrated to dryness to obtain 56.6g of foamy semisolid. 400mL volumes of water were added 10 times to provide a slurry. The slurry was filtered and washed with water. The solid was separated and dried to constant weight at 40°C under vacuum to produce 23.3g of preparation 18 as 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-oic acid) 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 0.1M HCl (2×1 L). The organic layer was washed with water (2×1 L) and then dried over MgSO4, 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 then added and the reaction was stirred for 17 hours. After the reaction was complete, EtOH was removed in vacuo and an orange oil (201 g) was left. The residue was dissolved in EtOAc (1.1 L) and washed with 0.1 M HCl solution (3 × 400 mL), then with NaHCO3 aqueous solution (400 mL). The layers were separated and the organic layer was then washed with saturated sodium chloride aqueous solution (1 × 400 mL). The organic matter was concentrated to produce an orange oil (~190 g). Acetone (400 ml) was added and the resulting suspension was then filtered to remove inorganics. The mixture was concentrated and then purified by normal phase chromatography (1.1 kg silica gel prepared with 60 / 40 heptane / acetone) and eluted with an eluent of increasing polarity (collecting ~3 L fractions). The components of at least 95 HPLC area % were combined and concentrated to provide a viscous yellow oil (70 g) of 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, e.g., 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, 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, 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 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, 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 give 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, the measured value is 4810.5036).

[0071] Step 1 Step 1: A feed solution of Preparation 25 (1.05 eq.) was prepared in 5 volumes of DMSO / ACN (90:10 v / v). A second feed solution of Preparation 26 was prepared in 20 volumes of DMSO / ACN (90:10 v / v). A third feed solution was prepared of PyOxim (1.5 eq.) in 3 volumes of ACN. A fourth stream of DIEA (4 eq.) in ACN was prepared. The first three streams were pumped into a mixer, and DIEA was mixed at the mixer outlet, and the mixture was pumped through another mixer and 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 the remaining PyOxim (1-cyano-2-ethoxy-2-oxoethylideneaminooxy-tripyrrolidinophosphonium hexafluorophosphate). 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 27 was collected and sent 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 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). The third feed solution was prepared from PyOxim (4.5 kg, 8.53 mol) in ACN (47.33 kg, 60.22 L) to produce a 0.132 M solution. DIEA was added as a neat liquid. 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 at 20° C. and mixed with neat DIEA (4.0 eq., 0.446 mL / min). The mixture was pumped through another mixer and 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 give 88.6 kg of product solution.

[0073] Nanofiltration is a membrane-based filtration method used to separate chemicals 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 <500Da).

[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 as waste. To maintain a constant volume in the NF tank, fresh solvent, i.e., 70:30 vol / vol DMSO / ACN, is continuously pumped in to match the resulting permeability. The Preparation 27 product solution purified by nanofiltration proceeds directly to Step 2.

[0075] A solution of Fmoc-protected Preparation 27 and diethylamine (1.34 kg) in DMSO / ACN (88.6 kg) were added to the reactor. The mixture was stirred at 20°C for 2 hours to obtain 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 are removed on the permeate side and collected or discarded as waste separately. This operation is continued until sufficient removal of unwanted impurities is obtained. In order to maintain a constant volume in the nanofiltration tank, fresh solvent, i.e., 70:30 volume / volume DMSO / ACN, is 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, de-Fmoc and nanofiltration).

[0076] Step 1 Example - Analyze the results.

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

[0078] Table A.1 shows the high resolution mass spectrometry 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 Measured Fmoc-protected Preparation 27 and Preparation 27 confirmed by mass accuracy 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] Step 2: A feed solution of Preparation 28 (1.15 eq.) was prepared in 10 volumes of DMSO / ACN (90:10 vol / vol). 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.) (5 wt% solution) in ACN. The solution of Preparation 27 from Step 1 as well as 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 enters 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 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), resulting in a solution of Preparation 28 (94.7 mg / mL, 0.0421 M) in 90:10 DMSO:ACN vol / vol. A second feed solution was prepared as PyOxim (3.0 kg, 5.69 mol) in ACN (11.81 kg, 15.03 L), resulting in 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) and PyOxim (2.1 equiv, 2.9 g / min) were pumped into the 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 process 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 the reactor for nanofiltration. The nanofiltration process produced Preparation 29 in DMF (98.85 L, 42.24 mg / mL, 4.18 kg, 0.974 mol, 73.1% yield by coupling, de-Fmoc and nanofiltration).

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

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

[0088] Table A.3 shows the high resolution mass spectrometry 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 confirmed the measured material products. The monoisotopic masses of the 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 Synthetic preparation from fragments Preparation 30 (SEQ ID NO: 30) and Preparation 29 (SEQ ID NO: 29) Product 31 (SEQ ID NO: 31).

[0091] Step 3 Batch Process 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 a 5.0 wt % solution of DIEA in DMF (114.0 mg, 5.70 mg DIEA, 0.0441 mmol, 1.8 equiv) and a 10.1 wt % solution of (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, as well as the Preparation 30 and DIEA streams were pumped into a mixer, and the streams were cooled and combined with the cooled HATU solution at the outlet of the mixer. 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 salt loading of 19% by weight (17 wt% sodium chloride in water, 0.5 wt% sodium bicarbonate in water) was prepared. The product solution of Preparation 31 in DMF was then pumped into a mixed product mixed suspension reactor (MSMPR) together 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 was prepared of HATU (1.15 kg, 3.02 mol) in ACN (10.4 kg) 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 a 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 temperature and collected over 42 hours to obtain 131.8 kg of product solution. The product solution was precipitated in two portions. A 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) was then added to the reactor together with a 17 wt% NaCl aqueous solution / 0.5 wt% NaHCO3 aqueous solution (34.4 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 hour before adding water (32.2 kg in total) in two portions. 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 part of the product solution was precipitated in a comparable manner. 17 wt% NaCl aqueous solution / 0.5 wt% NaHCO3 aqueous solution (29 kg) and DMF (13.55 kg) were combined in a reactor and cooled to no more than 20°C. The second part of the product solution in DMF (65.2 kg) was added to the reactor with 17 wt% NaCl aqueous solution / 0.5 wt% NaHCO3 aqueous solution (36.8 kg) over 1 hour, maintained at 20°C, to obtain the precipitation 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 time), 0.5 hours each time, and filtered. Two displacement water washes (64 kg each) were then 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, assuming 60% potency, 0.972 mol, assuming 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 give 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, 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, and the temperature was raised and maintained 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 -5°C to -18°C. The remaining cold MTBE (294.3 kg, 397.7 L) was added over 1.5 hours, maintaining -5°C to -18°C, to give the precipitate of Example 3. The slurry was adjusted to 0°C and maintained for >0.5 hours, then filtered in three portions. The combined wet cake was reslurried twice with MTBE (114.7 kg, 155 L) and filtered before the last 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, stirred for 2 hours, and then DMF was filtered out from the resin. The resin was then washed twice in total with DMF. The Fmoc protected resin was then deprotected using 20% ​​PIP / NMP. Sampling was performed after the last PIP / NMP treatment to verify Fmoc removal, and UV analysis confirmed that >99% of 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 build the peptide backbone.

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

[0098] Amino acid activation: A previously prepared 12% w / w Oxyma Pure / NMP solution was fed into 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 min before the solution was transferred 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 operations are 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 in preparation for the next coupling.

[0100] Coupling of Ile (12) and Aib (13): The coupling of Fmoc-Ile (12) and Aib (13) was carried out by 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 activated symmetrical anhydride species. As determined by HPLC analysis, an extended coupling stirring time (18 hours) was required to achieve reaction completion (<1% decoupling).

[0101] Lys(20)ivDe deprotection (Preparation 23): The fully protected 39 amino acid Lys(20)ivDde group on the resin Boc-Tyr(1)-Ser(39) peptide backbone was selectively deprotected. Deprotection was achieved using an 8% w / w solution of hydrazine hydrate in DMF with stirring for 4 hours at ambient temperature. The deprotection reaction was monitored by HPLC with an IPC limit of <1% for the remaining Lys(ivDde) ​​component after deprotection as the target. 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 as solids to a reactor, 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 the sampling was 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 of Example 4A: Prepare a cleavage mixture consisting of trifluoroacetic acid (TFA), triisopropylsilane (TIPS), dithiothioethanol (DTT), DCM and water. Cool the cleavage mixture to 15±5°C. The reagents are 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 then 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 keep the internal temperature of the crude solution at ≤5°C. The initial MTBE charge accounted for ~45% of the total MTBE charge. A soft precipitate formed near the end of the addition of MTBE, but it was easily redissolved into the solution. The precipitated solution was then recooled to an internal temperature of -15±5°C. The feed rate for the second MTBE addition was about 5-10 times the initial MTBE feed rate and accounted for ~55% of the total MTBE charge. During the addition, the internal temperature of the precipitated 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 another 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 45wt% 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): Example 4A crude product 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 the pre-equilibrated Kromasil100-10-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) buffer, which achieved 30% ACN for two column volumes, and prepared for elution by increasing the mixed concentration of ACN from 30% to 35% through one column volume. Tirezepatide was eluted from the column using 1.5% ACN per column volume until elution was complete. The eluent was fractionated and purity was determined using RP-HPLC. The column was regenerated by increasing ACN from 47% to 65% through one column volume and continuously flowing 65% ACN through 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 injections; however, only the main peak fractions are continued to be processed and no further circulation is performed. After completing the mainstream pooling, the concentration and purity of the intermediates 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 is loaded onto a pre-equilibrated Kromasil 100-10-C8 packed column. The column is 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 achieves a 20% ACN solution for two column volumes. Tirzepatide is eluted from the column using 3.5% ACN per column increase until elution is complete. The eluate is fractionated and the purity is determined using RP-HPLC. After elution, the column is regenerated by increasing ACN to 80% through one column volume and continuing to flow 80% ACN for three column volumes. Before the next injection sequence, the column is rebalanced using 20% ​​ACN for two column volumes.

[0109] Pool 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. Use the single injection standard to process and pool the loop injection; however, only the main peak fraction will continue to be processed and no further cycles will be performed. After the mainstream pooling is completed, the concentration and purity of the intermediates 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 into an Amberchrom CG300M column. The two fractions were eluted using mobile phase E (10% ammonium acetate in water, 5% IPA, pH 8) and mobile phase F (isopropanol). The fractions were analyzed for peptide content and those with <3 mg / mL were discarded. The concentrated pooled fractions were stored at 20° C. before precipitation. The IEX method started with 10.9 kg of RP2 to provide 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 then 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. In the first ~37% 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% MTBE charge. The temperature during the feed was maintained at <5°C. The resulting precipitation slurry was cold filtered (≤-10°C). The filter cake was then washed with MTBE. The filter cake was dried to LOD<2%.

[0112] Humidification 4A: Example 4A was humidified by passing wet nitrogen through the filter dryer. The humidity of the exhaust gas stream at the filter outlet was monitored every 60 minutes. Humidification continued until <0.5% MTBE and <0.2% IPA remained in the wet cake. After the humidification process was completed, the nitrogen flow was switched to allow dry nitrogen to flow through the pure product cake of Example 4A. Water and residual solvents in the material were sampled according to specific IPC targets, and drying with dry nitrogen continued until the desired target water content of 5-7% w / w was reached. A total of 12.9 kg of Example 4A was isolated with a peptide content purity of >95%. 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 completely replaced by 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 associated with the use of DMF was the coupling of Ile 12 to Aib 13, in which NMP was retained. In this example, 17.6 kg of Sieber resin was coupled to 92.2 kg of Preparation 23 peptide intermediate on resin in 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 Preparation 24 on resin. 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 in Example 4A to provide 24.4 kg of Example 4B, 69.5% HPLC purity and 52.6% yield; and 21.3 kg of Example 4B, 88.3% HPLC purity and 45.2% yield. The crude intermediate was refrigerated (-20°C) until purification.

[0116] Example 4B Purification: Crude Dissolution: The crude Tirzepatide Example 4B was charged to a dissolution vessel and dissolved in 1:1 acetonitrile:water solution to a final concentration of 25 g solid / L solution. The pH of the resulting solution was adjusted to 8.5-9.5 with ammonium hydroxide to initiate the conversion of the depsipeptide isomers (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 API: Reverse Phase Purification 1 (RP1): The RP1 process is essentially as described in Example 4A and can be used to convert the depsipeptide to the API. The RP1 purification process 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 produced 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 stand before proceeding to Reverse Phase Purification 2 (RP2).

[0118] Reverse Phase Purification 2 (RP2): Reverse Phase Purification 2 (RP2) uses essentially the same purification method as described in Example 4A, and uses 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 before tangential flow filtration.

[0119] Tangential Flow Filtration (TFF): TFF membrane was installed and rinsed with water. Ammonium acetate buffer was prepared using low endotoxin purified water, acetic acid and ammonium hydroxide. Isopropanol was then added to provide a 5:95 100mM NH4OAc pH 8.0:IPA buffer. The 17g / L RP2 solution of Example 4B was concentrated to ~125g / 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. 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 (403.9L@17g / L, 9.87kg 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 hours 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: Wet essentially as described in Example 4A and applied to 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 >97.7% HPLC purity and 88.4% peptide content. Overall yield based on Sieber resin loading = 46%.

[0122] Example 5 Continuous synthesis of preparations using convergent chemistry in flow 31 The synthesis of preparation 31 by peptide fragments uses both chemical batch method and the method of sequentially adding fragments in tubular flow reactor. The general method of synthesis involves coupling two fragments by mixing two solutions together with coupling agent in tubular reactor, followed by adding alkali, and the extra residence time in tubular reactor to remove the FMOC protecting group. This is ready for the coupling material to add the next fragment. Use a nanofilter with a membrane to remove excessive reagents, alkali and solvent between continuous coupling reactions, and the membrane is sized to retain peptides and penetrate low molecular weight impurities. Diafiltration is used to remove low molecular weight impurities fully before subsequent coupling steps. The description and analysis results of the examples derived from these conversions are included below.

[0123] HPLC was used to confirm the synthesis of Preparation 31 from Preparation 29 and Preparation 30. Analytical methods 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 spectrometry 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 the sequence. The method uses a chemoselective reaction of two unprotected peptide segments to produce a transient thioester-linked intermediate. The thioester-linked intermediate rearranges to provide a full-length ligated product with a native peptide bond at the ligation site. Those skilled in the art will appreciate that native chemical ligation techniques can be used 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 swollen in 100 mL DCM at 0 ° C for 20 min. 9-fluorenylmethyl hydrazine carboxylate (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 hydrazine carboxylate solution. The solution was then slowly added to the resin at 0 ° C. Stir at 0 ° C for about one hour and then warm it 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, then with DMF (2 x 100 mL), water (3 x 100 mL), DMF (3 x 100 mL), methanol (3 x 100 mL), and finally with DCM (3 x 100 mL). The resin was dried in a vacuum oven at 27°C for 16 hours. The resin loading was 0.74 mmol / g as determined by quantitative NMR.

[0128] Synthesis of peptide hydrazide (17-mer) (Preparation 33) SEQ ID NO: 32 Take hydrazine-CTC resin (1.01g, load value: 0.65mmol / g) in a 40mL reactor vessel and swell with 3×4mL DCM (30s each), then swell with 2×10mL DMF (20min each) on a peptide synthesizer. Fmoc-Ile-OH (0.919g, 2.60mmol, 4 equivalents) and HBTU (0.99g, 2.61mmol, 4 equivalents) are dissolved in 7mL DMF. DIPEA (0.91mL, 5.22mmol, 8 equivalents) is added to the amino acid solution, and the volume is made up to 10mL with DMF. The activated amino acid solution is added to the resin. The slurry is mixed with nitrogen for 8 hours. After 8 hours, the resin is washed with 5×10mL DMF, 5×10mL DCM, and dried for 12 hours. The load of the resulting resin is 0.54mmol / 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: 3 equivalents of amino acid, 3 equivalents of OXYMA and 3.3 equivalents of DIC were used for amino acid coupling. After each coupling and the last deprotection, the resin was washed with 5×9 mL DMF and 1 min N2 mixture. At the end of the peptide hydrazide synthesis, the resin was washed with DCM under N2 mixture. The resin was dried on the synthesizer.

[0131] Deprotection and cleavage: 25 mL of a cleavage mixture made 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 kept for half an hour and then centrifuged. The peptide precipitate was then washed twice with 150 mL MTBE and centrifuged. The peptide precipitate was dried in a vacuum oven at 27°C for 16 hours. After drying, 1.25 g of a 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 acylation of lysine.

[0133] Deprotection of ivDde: Hydrazine monohydrate (64% w / w) (1.98 g, 25.3 mmol) was diluted to 24.4 g with DMF and 20 g was added to the resin. The slurry was stirred with a stream of nitrogen. After about 2 hours, it was washed with 5 x 9 mL 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. This 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 5×10 mL DMF and 7×10 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 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 (2.42 g) and mixed on a rotary mixer for 3 hours. The resin was filtered and washed with 2×2.0 mL TFA. The filtrate was poured into 200 mL cold MTBE and the peptide was immediately precipitated. The filter flask was washed with 2×2 mL TFA and poured into cold MTBE. It was cooled to -20°C and kept for 30 min, then centrifuged. The peptide precipitate was washed twice with 240 mL 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. It was purified by RP-HPLC on a Kromasil 100-10-C8 10 μm column (30 mm×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 for the next 25 minutes, and 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 equiv) was added to the peptide hydrazide solution and allowed to stir at -15°C for 20 min. At the same time, 1 mL of 2,2,2-trifluoroethanethiol (TFET) was made up to 10 mL with 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 the in situ thiolysis of the peptidyl azide generated by Preparation 33.

[0137] The pH of the reaction mixture was adjusted to about 6.95 with 5N sodium hydroxide solution. The thiolysis of the peptidyl azide was allowed to continue 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 6M guanidine hydrochloride and 0.3M sodium dihydrogen phosphate (pH 7.0) is the ligation buffer used in native chemical ligation. All solutions are prepared in this ligation buffer. 350.4mg (0.174mmol) of peptide thioester preparation 35 (SEQ ID NO: 34) was dissolved in 50mL of ligation buffer. An 8.0mL portion of 0.5M 4-mercaptophenylacetic acid (MPAA) solution was added to the peptide thioester solution. The peptide containing N-terminal cysteine ​​(preparation 34 (SEQ ID NO: 33), 524.6mg, 0.178mmol, 1.03 equivalents) was dissolved in 48mL of ligation buffer in a 50mL centrifuge tube. The solution of preparation 34 was added to the thioester solution. The test tube was rinsed with 2×8mL of ligation buffer (approximately pH 7.0) and added to the reaction mixture. The pH of the reaction mixture was adjusted to about 7 with 5NNaOH 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×250 mm) at ambient temperature with a linear gradient of 10% acetonitrile in water for the first 4 minutes followed by 20-50% acetonitrile in water (0.1% acetic acid and titrated to pH 9.0) over a 28 minute purification period. Approximately 372 mg (44.3%) of 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 about 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 thereto. 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 thereto. 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) reference standard. The expected 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 value: 0.65 mmol / g) was taken in a 40 mL reactor vessel and swollen on a Symphony synthesizer with 3 x 10 mL DCM (30 s each) and then 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 (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-tetraazatricosane-53-oic acid (Preparation 17, 365 mg / mL in DMF) solution (3.91 mmol, 3.0 equiv). DIPEA (1.4 mL, 8.04 mmol, 6.1 equiv) was added to the above solution and the volume was made up to 19 mL with DMF. The solution was mixed on a rotary mixer at room temperature 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 by quantitative NMR to be 0.26 mmol / g. 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 last deprotection, the resin was washed with 5 × 9 mL DMF mixed with 1 min N2. At the end of the peptide hydrazide synthesis, the resin was washed with 7 × 10 mL DCM mixed with 1 min N2. The resin was then dried on the synthesizer for about 12 hours.

[0144] Deprotection and cleavage: 25 mL of a cleavage mixture made 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 (2×2.5 mL), and the filtrate was poured into 175 mL of cold MTBE. The filter flask was washed with TFA (2×2.5 mL), and the washes were poured into cold MTBE. It was cooled to -20°C and kept for 30 min, then centrifuged. The peptide precipitate was then washed twice with 150 mL of MTBE and centrifuged. The peptide precipitate was dried in a vacuum oven at 27°C for 16 hours. After drying, 1.70 g of crude peptide hydrazide preparation 39 (SEQ ID NO: 36) was obtained. The crude peptide hydrazide, Preparation 39, was purified by RP-HPLC at ambient temperature on a Waters XSelect CSH C18 10 μm column (10 mm×250 mm) with a linear gradient of 10% acetonitrile in water for the first 3 minutes, 20-55% acetonitrile in water for 23 minutes, and constant 0.1% TFA for 28 minutes. Approximately 110 mg of partially purified hydrazide Preparation 39 was obtained.

[0145] Deprotection and cleavage: 25 mL of a cleavage cocktail 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 2×2.5 mL TFA. The filtrate was poured into 200 mL cold MTBE and the peptide precipitated immediately. The filtrate flask was then washed with 2×2 mL TFA and the washes were poured into cold MTBE. It was cooled to -20°C for 30 min and then centrifuged. The peptide precipitate was then washed twice with 240 mL MTBE and centrifuged. The peptide precipitate was then dried in a vacuum oven at 27°C for 16 hours. 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) is the ligation buffer used in native chemical ligation. All solutions are prepared in this ligation buffer. Partially purified peptide hydrazide (preparation 39, 56mg, 0.019mmol) was dissolved in 5mL 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. 0.25mL of 1M sodium nitrite solution (0.25mmol, 13.2 equivalents) was added to the peptide hydrazide solution and stirred at -15°C for 10min. After 10min, 0.8mL of 0.5M 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 about 7.0 with 5N sodium hydroxide solution. The thiolysis of the peptidyl azide was carried out for 30min.

[0147] About 0.62 mmol of preparation 40 (SEQ ID NO: 37) was synthesized on Sieber amide resin using standard SPPS protocol. Preparation 40 (26.1 mg, 0.014 mmol, 0.74 quiv) containing N-terminal cysteine ​​was dissolved in 1 mL of connection buffer. The solution of preparation 40 was added to the thioester solution. The vial containing preparation 40 was rinsed with 1 mL of connection 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 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 SEQID 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 H-C-F-V-Q-W-L-I-A-G-G-P-S-S-G-A-P-P-P-S-NH2SEQ ID NO:38 SEQ ID NO:39 SEQ ID NO:40

Claims

1. A compound of SEQ ID NO: 10 or a pharmaceutically acceptable salt thereof: SEQ ID NO:10 wherein the compound is optionally bound to a resin, and wherein one or more of the Fmoc, Trt and / or Boc protecting groups may be independently replaced by an alternative protecting group.

2. A compound of SEQ ID NO: 16 or a pharmaceutically acceptable salt thereof: wherein the compound is optionally bound to a resin, and wherein one or more of the Fmoc, Trt and / or Boc protecting groups may be independently replaced by an alternative protecting group.

3. A compound of SEQ ID NO: 26 or a pharmaceutically acceptable salt thereof: wherein the compound is optionally bound to a resin, and wherein one or more of the Fmoc, Trt and / or Boc protecting groups may be independently replaced by an alternative protecting group.

Images