Process for preparing GIP / GLP1 dual agonists

Abstract

The present invention provides a process and intermediates for producing GIP / GLP1 dual agonist peptides, tilzepatide, or pharmaceutically acceptable salts thereof. [Solution] A process is provided for converting a depsipeptide isomer into a desired peptide, comprising: a. adjusting the pH of the depsipeptide isomer to approximately pH 7 to approximately pH 10; and b. incubating the depsipeptide isomer at pH 7 to pH 10 for at least 1 hour.

Description

Detailed Description of the Invention

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

[0002] Diabetes is a chronic disease characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both. In type 2 diabetes ("T2D"), the combined effects of insulin deficiency and insulin resistance are associated with elevated blood glucose levels. Tirzepatide, a GIP / GLP1 dual agonist, is described and claimed in U.S. Patent No. 9,474,780 ("the '780 patent"). Tirzepatide may be useful in the treatment of T2D.

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

[0004] There is a need for processes and intermediates that enable improved techniques for the production of tirzepatide with a combination of advantages including commercially desirable purity. Similarly, there is a need for an efficient and environmentally "green" process that includes stable intermediates for providing tirzepatide with fewer purification steps. Improved techniques are also needed to provide a tirzepatide manufacturing process that minimizes waste streams to enhance the safety of both the environment and the operator. The large-scale preparation of pharmaceutically superior tirzepatide presents several technical challenges that can affect overall yield and purity. There is a need for a process that avoids the use of transition metals and / or harsh reaction conditions that are incompatible with peptide synthesis.

[0005] The present invention aims to meet these needs by providing novel intermediates and processes useful for the production of tilsertide (SEQ ID NO: 1), or pharmaceutically acceptable salts thereof. The improved tilsertide production process of the present invention provides intermediate and process reactions that embody a combination of advancements, including an efficient route with fewer steps while maintaining high quality and purity. Importantly, the improved process and intermediates reduce resource intensity and minimize waste streams.

[0006] The improved processes described herein provide various embodiments of intermediates useful for the generation of tilsertide.

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

[0008] Compounds with the following formula: [ka] or provide a pharmaceutically acceptable salt thereof.

[0009] Compounds with the following formula: [ka] or provide a pharmaceutically acceptable salt thereof.

[0010] This invention provides a process for preparing tilzepatide using nanofiltration.

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

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

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

[0014] The peptide-lysine-NH2 bonded to the resin forms a compound with the following formula: [ka] The present invention provides a process for selectively acylating lysine amino acids, or their pharmaceutically acceptable salts.

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

[0016] This invention provides a process for converting depsipeptide isomers, adjusting the pH of the depsipeptide isomers to approximately 8.5 to 9.5.

[0017] This invention provides a process for converting a depsipeptide isomer, wherein the depsipeptide isomer is the compound of SEQ ID NO: 40 or a pharmaceutically acceptable salt thereof.

[0018] A radical-based desulfurization method is provided, comprising contacting a peptide with a radical initiator. In one embodiment, desulfurization comprises contacting a desulfurization-suitable peptide with a water-soluble radical initiator. In one embodiment, the radical initiator is an azo initiator. In one embodiment, the radical initiator is selected from the group consisting of 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (VA-044) and 2,2'-azobis(2-methylpropionamidine) dihydrochloride (VA-050).

[0019] The radical-based desulfurization method provided herein is environmentally desirable, transition metal-free, and suitable for peptide synthesis.

[0020] As used herein, the following abbreviations have the meanings set forth herein: "SPPS" means solid-phase peptide synthesis, "Fmoc" means fluorenylmethyloxycarbonyl chloride, "Pip" means piperidine, "DIC" means diisopropylcarbodiimide, "Oxyma" means ethylcyanohydroxyiminoacetate, "DCM" means dichloromethane, "IPA" means isopropanol, "MTBE" means methyl-tert-butyl ether, and "TFA" means trifluoroacetic acid. In this product, "TIPS" stands for triisopropylsilane, "DTT" stands for dithiothreitol, "UPLC" stands for ultra-high performance liquid chromatography, "HFIP" stands for hexafluoroisopropanol, "CTC" stands for chlorotrityl, "HATU" stands for (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate), "TFET" stands for 2,2,2-trifluoroethanethiol, and "DIEA" stands for N,N-di "AEEA" stands for sopropylethylamine, "TCEP" stands for 17-amino-10-oxo-3,6,12,15-tetraoxa-9-azaheptadecanoic acid, "DCU" stands for dicyclihexylurea, "DCC" stands for dicyclohexylcarbodiimide, "TMSA" stands for trimethylsilylamide, "HOBt" stands for hydroxybenzotriazole, "HRMS" stands for high-resolution mass spectrometry, and "LPPS" stands for liquid-phase peptide synthesis. "MPR" stands for Mixed Product Mixed Suspension Reactor, "MPA" stands for Mobile Phase A, "MPB" stands for Mobile Phase B, "L-GSH" stands for L-Glutathione Reduction Solution, "TZP" stands for Tylzepatide, "AP" stands for Pharmaceutical Active Ingredient, "API" stands for Active Pharmaceutical Ingredient, "PyBOP" stands for (Benzotriazole-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, "DEA" stands for Diethylamine, and "TBTU" stands for 2-(1H-Benzotriazole-1-yl)-1,1,3,"TNTU" means 3-tetramethylaminium tetrafluoroborate, "PyOxim" means 1-cyano-2-ethoxy-2-oxoethylideneaminooxy-tris-pyrrolidino-phosphonium hexafluorophosphate, and "PyClock" means 6-chloro-benzotriazole-1-yloxy-tris-pyrrolidinophosphonium hexafluorophosphate. As shown herein, single-letter abbreviations of amino acids are shown in bold, and atoms are shown as non-bold text, usually in a smaller font, to distinguish them from single-letter amino acid abbreviations. Where used herein, if an amino acid abbreviation appears with a number above the amino acid, the number refers to the position of the corresponding amino acid in the final tilzepatide product. The numbers are provided for convenience, and the appearance or absence of such numbers in a sequence does not affect the amino acid sequence or peptide represented in such a sequence. As used herein, the term “protected” means that a protecting group is attached to the indicated position. Those skilled in the art will recognize that various protecting groups are well known and that alternative protecting groups may be suitable for specific processes.

[0021] Those skilled in the art will understand that alternative resins exist for constructing the peptides presented herein. For example, Sieber amide resins and Rink amide resins are well known to those skilled in the art for preparing the peptides disclosed herein, but alternative resins may be selected for the preparation of the peptides described herein. For example, but not limited to, 2-CTC and related resins can be used to prepare the target peptide and subsequently perform the C-terminal amidation step.

[0022] Solid-phase peptide synthesis (SPPS) builds are performed using standard fluorenylmethyloxycarbonyl chloride (Fmoc) peptide chemistry techniques utilizing sequential coupling with an automated peptide synthesizer. After swelling the resin with DMF, deprotection is performed using 20% ​​piperidine (Pip) / DMF (3 x 30 minutes). For subsequent Fmoc deprotection, a 3 x 30 minute treatment with 20% Pip / DMF is used, and for more difficult couplings, a 4 x 30 minute treatment is used. After deprotection, the resin is washed with 10 times the volume of DMF washing solution for 5 x 2 minutes. For pre-activation of amino acids, a diisopropylcarbodiimide (DIC) / cyanohydroxyiminoethyl acetate (Oxyma) DMF solution is used at room temperature for 30 minutes. Coupling of activated amino acids to the resin-bound peptide occurs for the specified time for each individual amino acid. After each coupling, solvent washing with 10 volumes of DMF is performed for 5 x 2 minutes. To isolate the final product, the resin-bound product is washed with 10 volumes of DCM for 5x2 minutes to remove DMF. The resin is washed with 10 volumes of IPA for 2x2 minutes to remove DCM, then washed with 10 volumes of methyl-tert-butyl ether (MTBE) for 5x2 minutes, and the product is vacuum-dried at 40°C. The resin-bound product is stored refrigerated (-20°C). For analysis, the peptide is cleaved from the resin in the following ratio (0.93v / 0.04v / 0.03v / 0.03w) using an acidic cocktail consisting of trifluoroacetic acid (TFA) / H2O / TIPS (triisopropylsilane) / DTT (dithiothreitol). The resin is swollen with DCM (4-5 mL, 3x30 minutes) and drained. The cleavage cocktail (4-5 mL) is added to the pre-swollen resin, and the suspension is stirred at room temperature for 2 hours. The solution is filtered, and then the resin is washed with a small amount of DCM and mixed with the cleavage solution. Pour the resulting solution into 7 to 10 times its volume of cold (0°C) methyl tert-butyl ether (MTBE). Allow the suspension to mature at 0°C for 30 minutes, then centrifuge the resulting precipitate and decant the clear solution. Suspend the residue in an equal volume of MTBE, and centrifuge and decant the resulting suspension again. After decanting the clear MTBE solution of the precipitated peptide, vacuum dry overnight at 40°C.

[0023] Synthesis of preparation 1: Sequence ID 2 For synthesis, Fmoc-Sieber amide resin is used at a loading of 0.71 mmol / g. The general SPPS procedure is used with the following modifications. [Table 1] [Table 2]

[0024] Soft decomposition of preparation 1: Ten identical deprotection reactions are carried out in parallel on approximately 0.5 mmol scale resin-bound preparation 1 using the following protocol: 1) Add 1.55 g (approximately 0.5 mmol) of resin-bound preparation 1 to a 40 mL frit reactor. 2) Swell with 3 x 15 mL of DMF (15 minutes each). 3) Treat with 3 x 15 mL of 20% Pip / DMF (30 minutes each). 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 the five 40 mL reaction vials. 6) Add one-fifth of the resin-bound preparation 1 (2.75 g) to each TFA solution vial, cap the vial, and mix with a rotary wheel for 5 minutes. 7) Filter the mixture and wash with 100 mL of DCM to make a total volume of filtrate 500 mL. 8) Combine the filtrates and transfer to a round-bottom flask containing 1000 mL of MTBE. 9) Concentrate the resulting suspension into a pale yellow oil, grind 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 overnight in a vacuum oven at 33°C to produce 5.35 g (91% yield) of white solid. Analysis of the separated solid using UPLC (98.57 area%, combined t-Bu deprotection byproduct 0.99%).

[0025] Synthesis of preparation 2: Sequence ID 3 For synthesis, Fmoc-Gly-OH2-CTC resin is used with a loading of 0.61 mmol / g. The general SPPS procedure is used with the following modifications. [Table 3] [Table 4]

[0026] Soft cutting of Preparation 2: When Preparation 2 (3.06 g, 1.12 mmol) bound to the resin and 30 mL of 30% HFIP DCM solution are added to a 40 mL glass scintillation vial, the solution is observed to change to red. The vial is stirred by rotating it on a rotary wheel at ambient temperature for 1 hour. The resin is filtered out and washed with 3 x 10 mL of DCM. The solvent is removed under vacuum to form a glassy foam (35°C bath, 10 Torre, 2.34 g), which is then replaced with a small amount of IPA (24 mL), and water (24 mL) is added dropwise at room temperature over 25 minutes. The resulting solution is stirred for 30 minutes and then filtered. A 3 x 10 mL H2O cake is washed, and the washed cake is dried overnight in a vacuum oven at 25 Torre, 35°C. This produces Preparation 2 as a white solid (1.81 g).

[0027] Synthesis of preparation 4: Sequence ID 4 For synthesis, Fmoc-Leu-OH2-CTC resin is used with a load of 0.68 mmol / g. The general SPPS procedure is used with the following modifications. [Table 5] [Table 6]

[0028] Preparation 4 Soft Cutting: When Preparation 4 (2.0 g, 0.62 mmol) bound to the resin and 10 mL of 30% HFIP DCM solution are added to a 20 mL glass scintillation vial, the solution is observed to change to red. The vial is stirred by rotating it on a rotary wheel at ambient temperature, then the resin is filtered off, washed with 3 x 2 mL of DCM, and the solvent is removed under vacuum to form a glassy, ​​viscous foam. The foam is dissolved in 5.2 mL of DMSO. 6 mL of water is added to this solution at an equal flow rate (temperature approximately 15°C) over 45 minutes, followed by the addition of 1 mL of water. Once the peptide solution has been completely added, another 6 mL of water is added over 45 minutes. A white solid precipitate forms upon addition. The resulting slurry is stirred at 15°C for 30 minutes. The solid is filtered, washed with 6 mL of water, and then transferred to a vacuum oven at 35°C and 25 Torre. This yields preparation 4 (Boc-1-14-OH, 1.0763 g) as a white, fluffy solid.

[0029] Synthesis of preparation 3 using LPPS: Sequence ID 5 Add Preparation 2 (500 mg, 0.183 mmol), Preparation 1 (179 mg, 0.175 mmol), and DMSO (10 mL) to a 20 mL glass scintillation vial. Add DIEA (46 μL, 0.265 mmol) to this solution, followed by PyBOP (benzotriazole-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate) (123 mg, 0.230 mmol). Stir the reaction mixture for 2 hours, then add diethylamine (DEA) (183 microliters, 1.77 mmol), and stir the resulting solution for 2 hours. Transfer the reaction contents to a syringe and add to a stirred 50 mL flask, simultaneously adding water (12 mL) dropwise over 1 hour. Once the addition is complete, collect the precipitated product by filtration and wash with water (2 x 4 mL). The wet cake was dried under vacuum at 35°C for 18 hours to obtain preparation 3 as a white solid (0.6003 g, 88% yield, C 184 H 261 N 31 O 38(HRMS calculation predicted value: 3512.9444, actual value: 3512.9430).

[0030] Synthesis of preparation 5 using LPPS: Sequence ID 6 [ka]

[0031] Add Preparation 3 (338.8 mg, 0.091 mmol), Preparation 4 (192.1 mg, 0.091 mmol), and DMSO (10 mL) to a 20 mL glass scintillation vial. Add PyBOP (63.5 mg, 0.118 mmol), followed by DIEA (79 microliters, 0.454 mmol) to this solution. Stir the reaction mixture for 2.5 hours. Inject the reaction contents into a syringe and add the contents to a stirred 50 mL flask, simultaneously adding water (12 mL) dropwise over 1 hour. After the addition is complete, collect the precipitated product by filtration and then wash with water (2 x 4 mL). Dry the wet cake under vacuum at 35°C for 18 hours to obtain Preparation 5 as a white solid (0.3568 g, 70% yield, C 293 H 435 N 45 O 64 (HRMS calculation predicted value: 5608.2168, actual value: 5608.2066).

[0032] Synthesis of preparation 6 by Method 1 (LPPS) [ka] [ka]

[0033] Eicosanedioic acid, mono(1,1-dimethylethyl) ester (15.0 kg, limiting reactant), and N-hydroxysuccinimide (1.2 equivalents) are dissolved in ethyl acetate at 27°C. A solution of DCC (1.25 equivalents) dissolved in ethyl acetate is added, and the reaction mixture is stirred at 22°C for 24 hours. The resulting DCU byproduct is removed by filtration, and the organic phase is extracted three times with 5% NaCl aqueous solution. After extraction, the organic phase is concentrated, co-evaporated with isopropanol, and crystallized by adding heptane. After filtration, the filter cake is rinsed with heptane and dried at 25°C to obtain 17.0 kg of INT1 in 87% yield and 99% purity.

[0034] H-Glu-OtBu (7.7 kg, 1.1 equivalents) was dissolved in DCM (54 L) at 20°C, then a solution of TMSA (11.3 kg) dissolved in DCM (7 L) was added, and the reaction mixture was stirred at 40°C for 1 hour. INT1 (17.0 kg) DCM solution was added at room temperature and stirred for 8 hours. After the reaction was complete, DCM was replaced with ethyl acetate by distillation. The organic phase was washed three times with a 2% aqueous solution, then four times with a KHSO4 / NaCl aqueous solution along with a 2% NaCl aqueous solution. After separating and removing the aqueous phase, the organic phase was concentrated with isopropanol, diluted with isopropanol, and crystallized by adding water. After filtration, the filter cake was washed with a water / isopropanol mixture and dried at 30°C to produce 17.3 kg of INT2 in 86% yield and 99% purity.

[0035] INT2 (17.3 kg) and N-hydroxysuccinimide (4.1 kg, 1.2 equivalents) were dissolved in ethyl acetate (336 kg) at 27°C. A solution of DCC (8.33 kg, 1.25 equivalents) was added to the ethyl acetate, and the reaction mixture was stirred at 22°C for 24 hours. The resulting DCU byproduct was removed by filtration. The organic phase was concentrated and co-evaporated with isopropanol, and then crystallized by cooling the isopropanol solution (approximately 125 L). The filtered cake was then rinsed with cold isopropanol and dried at 25°C to obtain 16.3 kg of INT3 in 81% yield and 96% purity.

[0036] 17-amino-10-oxo-3,6,12,15-tetraoxa-9-azaheptadecanoic acid (AEEA2) (8.1 kg, 26.3 mol) is suspended in DCM (54 L) at 22°C, and TMSA (7.68 kg, 59.9 mol) is dissolved in DCM (6.2 L). The reaction mixture is stirred at 40°C for 1 hour. INT3 (16 kg) is suspended in DCM (31 L) at 35°C and added to the TMS-protected (AEEA2) mixture at 22°C. The reaction mixture is stirred for 12 hours. After the reaction is complete, the mixture is concentrated and then replaced with ethyl acetate. The organic phase is washed three times with 2% aqueous solution. The mixture is washed four times with KHSO4 / NaCl aqueous solution (approximately 200 L), then with 2% NaCl aqueous solution (approximately 200 L) to reach a target pH of 4.5. The organic phase is concentrated and replaced with acetonitrile. The acetonitrile solution was cooled to -20°C, and the resulting suspension was aged at -20°C for 15 hours. The mixture was filtered, the filtered cake was rinsed with cold acetonitrile, and dried below 0°C to obtain 18.4 kg of Preparation 6 (88% yield) with 96% purity. Overall yield = 53%.

[0037] Synthesis of preparation 6 by Method 2 (SPPS) Alternatively, preparation 6 may be prepared using solid-phase peptide synthesis with a peptide synthesizer.

[0038] Use the standard coupling procedure.

[0039] Standard coupling conditions: 0.133M, 2.0 equivalents of HATU, 5.0 equivalents of DIEA, ambient temperature, 3 hours, deprotected with 20% piperidine / DMF for 3 x 15 minutes.

[0040] Resin filling: FmocNH-AEEA (0.99 mmol / g) on ​​2-CTC resin: 1.01 g in each parallel reaction.

[0041] An automated program using DMF swelling, followed by Pip / DMF; DMF washing; amino acid, DIEA, HATU mixing; and a DMF washing cycle followed by drying.

[0042] The resin is cut by stirring the combined lot in 30% HFIP / DCM (240 mL) for 1.5 hours. The resin is filtered and washed, and the solvent is removed from the filtrate under vacuum. The resulting oil is dissolved in acetonitrile, and the solvent is removed again. This operation yields 30.47 g (146% of the theoretical yield) of a viscous yellow oil, which contains 52.3 area% of the desired product by ULC analysis. The crude product is purified by flash chromatography (elution with 500 g of silica gel, 85% DCM / 10% methanol / 5% acetic acid, and a fraction of 38 × 100 mL is collected). The previously chromatographic concentrate (17.94 g) is crystallized to obtain 13.4 g (74.7% yield) with a ULC purity of 91.65 area%.

[0043] Example 1 [ka] [ka]

[0044] Synthesis Example 1 Sequence ID 1 Into the first HPLC vial, add Preparation 5 (10.5 mg, 0.00187 mmol) and DCM (200 μL, 20 L / kg). To this solution, add 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). Stir the solution at 24 °C for 1 hour to obtain an unisolated solution of Preparation 7 (SEQ ID NO: 7). Into the second HPLC vial, add DCM (150 μL), followed by Preparation 6 (0.118 M in DCM, 16 μL, 0.00189 mmol), PyBOP (0.186 M in DCM, 16 μL, 0.00298 mmol) and DIEA (0.573 M in DCM, 5 equivalents). Add the contents of the second vial to the first vial and stir the reaction mixture for 1 hour to obtain an unisolated solution of Preparation 8 (SEQ ID NO: 8). Concentrate the solution of Preparation 8 under vacuum and add 50 μL of a solution of trifluoroacetic acid (4.65 mL), triisopropylsilane (20 μL) and DTT (20 mg) to the resulting solid. Stir the slurry for 18 hours and monitor by HPLC to confirm the formation of Example 1 (C 225 H 348 N 48 O 68 The predicted HRMS calculated value for is 4810.5249, the measured value is 4810.5257).

[0045] Synthesis of Preparation 9 SEQ ID NO: 9 Sieber amide resin (13.42 g, 0.75 mmol / g, 10.1 mmol) is suspended in DMF (130 mL, 10 vol) for about 20 minutes and then drained. Wash the resulting resin with DMF (80 mL, 6 vol) for about 5 minutes. Treat the Fmoc amino acid resin twice with a 5 vol% piperidine, 1.25 vol% DBU, 1.0 wt% HOBt / DMF solution (80 mL, 6 vol) for 10 minutes and 20 minutes respectively to remove the Fmoc group. After draining the de-Fmoc solution, wash twice with DMF (80 mL, 6 vol), twice with MTBE (80 mL, 6 vol), and again twice with DMF (80 mL, 6 vol).

[0046] Standard Fmoc chemistry is used to assemble the amino acid chain. Generally, 1.5 equivalents of Fmoc-amino acids and HOBt (2.47 g, 20% wet, 14.6 mmol, 1.46 equivalents) are dissolved in DMF (60 mL, 4.5 vol), followed by the addition of DIEA (1.94 mL, 11.1 mmol, 1.11 equivalents). The resulting solution is cooled to below 5°C in an ice bath and activated by adding TBTU (4.83 g, 15.0 mmol, 1.5 equivalents). The mixture is left to stand at 0°C to 5°C for about 5 minutes. DCM (60 mL, 1.5 vol) is added to the resin, followed by the addition of the activated Fmoc-amino acid solution. The resulting mixture is stirred at near ambient temperature for 2 hours. The de-Fmoc procedure is repeated, and the remaining amino acids are coupled sequentially. After the final Fmoc removal procedure is complete, the resin is washed twice with 2-propanol (130 mL, 10 vol) for 5 minutes, followed by six washes with MTBE (130 mL, 10 vol). The resin is vacuum-dried at 35°C to obtain the preparation 9-Seiber (21.21 g, 0.435 mmol / g theoretical, 91.7% yield based on mass increase).

[0047] A portion of the resin composite (10.15 g, 0.435 mmol / g, 4.41 mmol) of Preparation 9 is treated with a washing step of 5 vol% TFA and DCM in a DCM (101 mL, 10 vol) solution. The cut fraction and washing solution are neutralized with DIEA (26.29 g, 35.5 mL, molar ratio of 1.01:1 to TFA). The fractions are combined and concentrated under vacuum to 50% of the original volume. The DCM solution is washed with saturated NaHCO3 aqueous solution (2 × 94 mL). The resulting solution is dried on anhydrous MgSO4 and concentrated to dryness until a gum-like solid is obtained. This gum-like solid is reslurried with MTBE (100 mL) below 5°C to decompose the gum and obtain a white slurry product. The slurry of the white powder obtained in preparation 9 is filtered, washed, and dried to obtain a white powder (3.84 g, 92.3 area%, 37.8 wt% DIEA·TFA, 57.4 wt%, 2.29 mmol, 51.9% yield, C 46 H 78 N 10 O 12 (HRMS calculation predicted value: 962.5801, actual value: 962.5806).

[0048] Synthesis of preparation 10 Sequence ID 10 The Fmoc-Gly-Gly-O-2CTC resin composite (18.09 g, 0.57 mmol / g, 10.3 mmol) is suspended in DMF (180 mL, 10 vol) for 20 minutes, then drained. The resulting resin is washed with DMF (108 mL, 6 vol) for 5 minutes. The Fmoc amino acid resin is treated twice with a solution of 5 vol% piperidine, 1.25 vol% DBU, and 1.0 wt% HOBt / DMF (108 mL, 6 vol) for 10 minutes and 20 minutes, respectively, to remove the Fmoc groups. The de-Fmoc solution is drained, and the resin is washed twice with DMF (110 mL, 6 vol), twice with MTBE (110 mL, 6 vol), and twice with DMF (110 mL, 6 vol). Chain assembly is performed according to standard Fmoc chemistry.

[0049] For amino acid coupling, generally, 1.5 equivalents of Fmoc-amino acids and HOBt (2.54 g, 20% wet, 15.0 mmol, 1.5 equivalents) are dissolved in DMF (80 mL, 4.4 vol), followed by the addition of DIEA (1.94 g, 15.0 mmol, 1.5 equivalents) to induce amino acid coupling. The resulting solution is cooled to 0-5°C in an ice bath and activated by adding 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU) (4.84 g, 15.1 mmol, 1.5 equivalents). The mixture is left to stand at 0-5°C for 5 minutes. Then, DCM (35 g, 1.5 vol) is added to the resin, followed by the addition of the activated Fmoc-amino acid solution. The resulting mixture is stirred at room temperature for 2 hours. After the synthesis steps were completed, the peptide resin was washed twice with 2-propanol (180 mL, 10 vol) for 5 minutes, then washed six times with MTBE (180 mL, 10 vol each), and dried at 35°C to obtain a 10-resin composite (25.52 g, 0.216 mmol / g, 53.6% yield).

[0050] A portion of the resin composite (10.075 g, 0.216 mmol / g, 2.18 mmol) of preparation 10 was treated three times with 1 vol% TFA in a DCM (100 mL, 10 vol) solution, and washed with DCM (75 mL, 7.5 vol). The cleaved fraction was neutralized and washed with pyridine (3.18 g, molar ratio of 1.01:1 to TFA). The fractions were combined and concentrated under vacuum and dried at 35°C or below. Reconstitution was performed with ethanol (40 mL, 10% vol of the combined filtrate), followed by concentration to dryness. Finally, the peptide was ground with stirring in deionized water (150 mL, 40% vol of the combined filtrate). The solid crude peptide precipitate was collected by centrifugation and washed twice with deionized water (150 mL each time). The solid was washed twice with n-heptane (100 mL each time), isolated, and dried under vacuum at 40°C to obtain preparation 10 (SEQ ID NO: 10) as a crisp, pale yellow solid (4.10 g, 72.4 area%, 3.0 wt% pyridine TFA, 70.2 wt%, 1.85 mmol, 85.1% yield, C 88 H 103 N 11 O 15 (HRMS calculation predicted value: 1553.7635, actual value: 1553.7656).

[0051] Synthesis of preparation 11 Sequence ID 11 H-alanine-O-2CTC resin composite (40.39 g, 0.5 mmol / g, 20.20 mmol) is suspended in DMF (400 mL, 10 vol) for approximately 20 minutes, then drained. The resulting resin is washed twice with DMF (400 mL, 10 vol) for 5 minutes each. Amino acid chain assembly is performed using standard Fmoc chemistry. Generally, 1.5 equivalents of Fmoc-amino acids and HOBt (5.51 g, 80 wt%, 32.6 mmol, 1.6 equivalents) are dissolved in DMF (150 mL, 3.7 vol), followed by the addition of DIEA (4.22 g, 32.7 mmol, 1.6 equivalents). The resulting solution is cooled to below approximately 5°C in an ice bath, and TBTU (10.39 g, 32.4 mmol, 1.6 equivalents) is added to activate it. The mixture is stirred at 0-5°C for approximately 5 minutes. Add DCM (80 mL, 2 vol) to the resin, followed by the activated Fmoc-amino acid solution. Stir the resulting mixture at approximately ambient temperature for 2 hours.

[0052] The Fmoc amino acid resin is treated twice with 5 vol% piperidine, 1.25 vol% DBU, and 1.0 wt% HOBt / DMF solution (240 mL, 6 vol) for 10 minutes and 20 minutes respectively to remove the Fmoc groups. The de-Fmoc solution is drained, and the resin is washed twice with DMF (240 mL, 6 vol), twice with MTBE (240 mL, 6 vol), and twice with DMF (240 mL, 6 vol). After the completion of the synthesis steps, the peptide resin is thoroughly washed twice with 2-propanol (400 mL, 10 vol) and six times with MTBE (400 mL, 10 vol each), then dried under vacuum at 35°C to obtain a packed resin with the last amino acid removed (74.82 g, 0.159 mmol / g, 11.90 mmol, 58.9% yield). The final amino acid, Fmoc-Leu-OH, is added separately to a portion of the resin (13.61 g, 0.159 mmol / g, 2.16 mmol). This resin is swollen with DMF (130 mL, 10 vol, 3 times) for at least 5 minutes each time, and then deprotected for 10 minutes and 20 minutes (twice with a 130 mL, 10 vol deprotection mixture prepared by dissolving 5.6 g piperidine, 1.67 g DBU, and 1.3 g HOBt in 120 mL of DMF). The resin is washed with DMF (80 mL, 6 vol, 2 times), MTBE (80 mL, 6 vol, 2 times), and DMF (80 mL, 6 vol, 2 times) for 5 minutes each time. Dissolve in DMF (50 mL, 3.7 vol), then add DIEA (0.54 g, 4.2 mmol, 1.9 equivalents) for coupling Fmoc-Leu-OH, Fmoc-Leu-OH (1.47 g, 4.16 mmol, 1.9 equivalents), and HOBt (0.704 g, 80 wt%, 4.17 mmol, 1.9 equivalents). Cool the resulting solution to below 5°C in an ice bath, activate with TBTU (1.34 g, 4.17 mmol, 1.9 equivalents), and stir at 0-5°C for 5 minutes. Add DCM (20 mL, 1.5 vol) to the resin, then add the activated Fmoc-amino acid solution. Stir the resulting mixture at near ambient temperature for 2 hours. Wash the resin with DMF (180 mL, 13 vol, twice), MTBE (180 mL, 13 vol, twice), and DMF (180 mL, 13 vol, twice) for 5 minutes each.The resin was washed with DCM (130 mL, 10 vol, 6 times, 5 minutes each), and then dried under vacuum at 35°C to obtain the filled resin (12.90 g, 0.203 mmol / g, 2.62 mmol, 121% yield).

[0053] A portion of the resin (7.09 g, 0.203 mmol / g, 1.44 mmol) was treated three times with 1 vol% TFA in DCM solution (70 mL, 10 vol) at near ambient temperature for 10 minutes each time, and then washed with DCM (55 mL, 7.5 vol). The cleaved fraction was neutralized and washed with pyridine (3.02 g, molar ratio of 1.02:1 to TFA). The fractions were combined and concentrated under vacuum and dried at 35°C or below. Reconstitution was performed with ethanol (28 mL, 11% vol of the combined filtrate), followed by concentration to dryness. Finally, the peptide was stirred in deionized water (105 mL, 40% vol of the combined filtrate). The solid crude peptide precipitate was collected by filtration and washed with deionized water (4 x 50 mL). The solid was washed with n-heptane (3 x 100 mL), isolated, and dried under vacuum at 40°C to obtain 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, C 127 H 192 N 14 O 28 The HRMS calculated predicted value was 2361.4031, and the measured value was 2361.4021. The overall yield of preparation 11 on the resin was 71.3%.

[0054] Synthesis of preparation 12 Sequence ID 12 The Fmoc-Aib-O-CTC resin composite (19.16 g, 0.54 mmol / g, 10.35 mmol) was suspended in DMF (190 mL, 10 vol) for 20 minutes and then drained. The resulting resin was washed with DMF (190 mL, 10 vol) for 5 minutes and then drained. Piperidine (77.82 g), DBU (23.16 g), HOBt (18.09 g, 80 wt%), and DMF (1800 mL) were mixed to obtain a deprotection solution of 5% piperidine, 1.25% DBU, and 1.0% HOBt / DMF. The Fmoc-amino acid resin was treated twice with the deprotection solution (190 mL, 10 vol) for 10 minutes and 20 minutes, respectively, to remove the Fmoc groups. Drain the Fmoc-free solution, and wash the resin twice with DMF, twice with MTBE, and twice with DMF (190 mL, 10 vol for each wash).

[0055] Add DIEA (2.62 g, 20.3 mmol, 2.0 equivalents) to a solution of Fmoc-Ile-OH (7.11 g, 10.1 mmol, 2.0 equivalents) in DMF (85 mL). Cool the resulting solution to 0-5°C, add 6-chloro-benzotriazole-1-yloxy-tris-pyrrolidinophosphonium hexafluorophosphate (PyClock) (11.36 g, 20.06 mmol, 2.0 equivalents), and dissolve completely. After standing for 3-5 minutes, add the activation solution to the H-Aib-O-CTC resin composite pre-swollen with DCM (30 mL, 1.5 vol). Raise the temperature to ambient temperature and stir for 2 hours. As shown in the assay evaluation, approximately 18% of the material remains unreacted. Wash twice with DMF, twice with MTBE, and twice with DMF (190 mL, 10 vol each). A solution of Fmoc-Ile-OH (10.63 g, 30.08 mmol, 6 equivalents) dissolved in DMF (165 mL) is added to Oxyma (50 mL, 0.6 M in DMF, 30 mmol, 6 equivalents) and DIC (50 mL, 0.66 M in DMF, 33 mmol, 6.6 equivalents). The mixture is stirred at approximately ambient temperature for 5 minutes, then added to the resin and stirred for 18 hours. A mixture of pyridine, acetic anhydride, and DMF is added to the resin and stirred for 0.5 hours. The resin is washed with DMF (5 x 140 mL, 7 vol), then DMF (2 x 180 mL, 9 vol), MTBE (2 x 180 mL, 9 vol), and then DMF (2 x 180 mL, 9 vol).

[0056] For the remaining amino acids, the rest of the chain assembly is carried out sequentially using standard Fmoc chemistry. Generally, Fmoc-amino acids (2.0 equivalents) and HOBt (3.42 g, 80 wt%, 2.0 equivalents) are dissolved in DMF (85 mL), followed by the addition of DIEA (2.64 g, 2.0 equivalents). The resulting solution is cooled to 0-5°C in an ice bath, activated by adding TBTU (6.45 g, 2.0 equivalents), and left to stand at 0-5°C for 3-5 minutes. DCM (30 mL) is added to the resin, followed by the addition of the activated Fmoc-amino acid solution. The resulting mixture is stirred at room temperature for 2 hours. The resulting resin is washed twice with DMF, twice with MTBE, and twice with DMF (190 mL, 10 vol for each wash). The Fmoc-amino acid resin is treated twice with deprotection solution (190 mL, 10 vol) for 10 minutes and 20 minutes, respectively, to remove the Fmoc groups. After draining the Fmoc-free solution, wash the resin twice with DMF, twice with MTBE, and twice with DMF (190 mL, 10 vol each time).

[0057] The tetramer Boc-Y-Aib-E(tBu)G-OH (12.25 g, 2.0 equivalents) in DMF (50 mL) is activated with Oxyma (0.6 M in DMF, 30 mL, 20 mmol, 2 equivalents) and DIC (0.66 M in DMF, 33 mL, 22 mmol, 2.1 equivalents) for 5 minutes, and the last four amino acids are added as a tetramer. This mixture is added to the resin and coupling is performed for 18 hours. After 18 hours, the mixture is drained and the resin is washed with DMF (190 mL each, 5 minutes, 5 times). The tetramer (6.21 g, 1.0 equivalent) is added to DMF (40 mL), activated with PyBOP (5.77 g, 1.1 equivalents) and DIEA (3.32 g, 2.6 equivalents) for 5 minutes, and then this mixture is added to the resin and stirred for 4 hours. After 4 hours, the mixture is drained and washed with DMF (190 mL each, 5 times for 5 minutes). A mixture of DMF (105 mL), pyridine (13.48 g, 17 equivalents), and acetic anhydride (14.27 g, 14 equivalents) is added, the resin is capped, and the mixture is stirred for 1 hour. After chain assembly is complete, the peptide resin is washed with DMF 5 times (190 mL each) for 5 minutes each, and with DCM 6 times (190 mL each), then dried under vacuum at 35°C to obtain Preparation 12 resin complex (31.03 g, 0.2595 g / mmol theoretical value, 8.05 mmol, yield 77.8%). A portion of the resin composite (15.975 g, 0.2595 mmol / g, 4.146 mmol) of preparation 12 was treated three times with 1 vol% TFA in DCM solution (160 mL, 10 vol) at ambient temperature for 10 minutes each time, and then washed with DCM (120 mL, 7.5 vol). The cleaved fraction was neutralized and washed with pyridine (4.74 g, molar ratio of 0.94:1 to TFA). The fractions were combined and concentrated under vacuum and dried at 35°C or below. Reconstitution was performed with ethanol (30 mL, 5% vol of the combined filtrate), followed by concentration to dryness. The peptide was mechanically stirred with deionized water (242 mL, 40% vol of the combined filtrate) for 10 minutes. The solid crude peptide was collected by filtration and washed with deionized water (4 x 100 mL). The solid was washed with n-heptane (4 x 100 mL), isolated, and dried under vacuum at 35°C to obtain 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, C 103 H165 N 13 O 26 (HRMS calculation predicted value: 2000.1989, actual value: 2000.1968).

[0058] Synthesis of preparation 13 Sequence ID 13 Add Preparation 9 (2.887 g, 70.2 wt%, 1.30 mmol), Preparation 10 (3.576 g, 57.4 wt%, 2.13 mmol, 1.63 equivalents), DMSO (18.1 g, 16.4 mL), DMF (15.8 g, 16.7 mL), and DIEA (655 mg, 5.07 mmol, 3.89 equivalents) to a flask under N2 and stir until a golden solution is obtained. Cool the solution in ice water before adding PyBOP (1.414 g, 2.72 mmol, 2.08 equivalents). Remove the ice bath and allow the mixture to rise to ambient temperature. Monitor the reaction for about 5 hours to ensure proper conversion. Add the aliquot of diethylamine (2.116 g, 28.9 mmol, 22.2 equivalents) to the reaction mixture at ambient temperature. The mixture is stirred for approximately 1 hour to obtain a conversion of over 99% to Preparation 13. The product is precipitated by adding a mixture below 4°C containing saturated NaHCO3 aqueous solution (50 mL) and deionized water (50 mL) to the reaction mixture. The mixture is stirred under low temperature conditions for at least approximately 15 minutes. The muddy white slurry is filtered. The wet cake is washed with deionized water (3 × 50 mL) followed by MTBE (6 × 50 mL), and then dried at 40°C under vacuum with N2 purging for approximately 62 hours. This process yields Preparation 13 (4.45 g, 60.4 area%, 16.4 area% dibenzoflubene, 1.18 mmol, 90.5% yield, C 119 H 169 N 21 O 24 The HRMS calculated predicted value (2276.2649) and the measured value (2276.2550) are obtained as a pale yellow solid.

[0059] Synthesis of preparation 14 Sequence ID 14 Aliquotes of Preparation 11 (3.012 g, 48.7 wt%, 0.621 mmol, 1.00 equivalent) are placed in a flask under N2 together with Preparation 13 (3.951 g, 60.4 wt%, 1.05 mmol, 1.69 equivalents), DMSO (9.8 g, 8.9 mL), DMF (52.0 g, 55.0 mL), and DIEA (372 mg, 2.88 mmol, 4.63 equivalents). The mixture is stirred until a golden solution is obtained. The mixture is cooled to below 10°C using ice water. Aliquots of PyBOP (742 mg, 1.42 mmol, 2.30 equivalents) are added to the mixture. The ice bath is removed, and the mixture is allowed to rise to approximately ambient temperature. The reaction is monitored for conversion to Preparation 14 for approximately 22 hours. This results in a conversion of over 96%. If the temperature is below 10°C, add piperidine (530 mg, 6.22 mmol, 10.0 equivalents) to the cooled reaction mixture. Stir the mixture at ambient temperature for about 2 hours to obtain more than 99% conversion to preparation 14. Add the reaction mixture to another flask containing 0.5 N aqueous HCl solution (12.72 g, 6.23 mmol, 10.0 equivalents) and deionized water (16.71 g) below 4°C to obtain a precipitate of preparation 14. Stir the cold slurry for about 15 minutes and filter the white slurry. Wash the wet cake with deionized water (2 x 30 mL), saturated aqueous NaHCO3 solution (2 x 30 mL), deionized water (3 x 30 mL), and MTBE (4 x 45 mL), then dry at 40°C under vacuum with N2 purging for about 17 hours. Product, preparation 14, is obtained as a white powder (5.418 g, 48.9 area%, 0.603 mmol, 97.0% yield, C 231 H 349 N 35 O 49 (HRMS calculation predicted value: 4397.5893, actual value: 4397.6057).

[0060] Synthesis of preparation 15 Sequence ID 15 Add an aliquot of Preparation 12 (671 mg, 82.1 wt%, 0.275 mmol, 1.23 equivalents) to a flask under N2. Add Preparation 14 (2.009 g, 48.9 area%, 10.7 area% isomer, 0.223 mmol, 1.00 equivalent), DMSO (11.1 g, 10.0 mL), DMF (19.0 g, 20.1 mL), and DIEA (76 mg, 0.588 mmol, 2.63 equivalents) to the flask with stirring to obtain a golden-colored solution. Add an aliquot of 0.6 M HOAt (619 mg, 0.384 mmol, 1.72 equivalents) before cooling to -5°C. Add a sample of PyClock (220 mg, 0.397 mmol, 1.78 equivalents). The mixture is heated to near ambient temperature, resulting in approximately 84% conversion to Preparation 15. The reaction mixture is added to ice-cold deionized water (548 mL) over 10 minutes to isolate the product and allow it to precipitate. The reaction flask is rinsed with DMF (5 mL) and added to the slurry. The slurry is stirred for approximately 15 minutes, heated to near ambient temperature, and filtered. The wet cake is washed with deionized water (3 x 80 mL), and the white waxy solid is dried under vacuum at 35°C for 3.5 days to obtain Preparation 15 as a white powder (2.506 g, 41.6 area%, 0.163 mmol, 73.1% yield, C 334 H 512 N 48 O 74 (HRMS calculation predicted value: 6379.7777, actual value: 6379.8652).

[0061] Example 2 Synthesis of Example 2 Sequence ID 1 Add the TFA sample (19.656 g, 13.03 mL) to a flask under N2 conditions along with DCM (815 mg, 0.62 mL), DTT (434 mg), and TIPS (362 mg, 0.47 mL). Cool the mixture in ice water before adding water (468 mg, 0.47 mL). Add the Preparation 15 sample (1016 mg, 39.0 area%, 0.0620 mmol) to the mixture at 2°C to bring the solution. Warm the mixture to near ambient temperature and stir for about 2 hours. Add the reaction mixture to MTBE (150 mL) at -15°C and rinse the reactor with MTBE (3 mL). After about 10 minutes, centrifuge the slurry and decant the supernatant. Reslurry the wet cake with MTBE (3 x 50 mL), centrifuge after each wash, and decant the supernatant. The wet cake was dried under vacuum at 35°C to obtain Example 2 as a white solid (784 mg, 26.5 area%, 0.0432 mmol, 69.7% yield, C 225 H 348 N 48 O 68 (HRMS calculation predicted value: 4810.5249, actual value: 4810.5642).

[0062] Synthesis of preparation 16 Sequence ID 16 In the synthesis, Fmoc-Gly-OH2-chlorotrityl resin is used at a loading of 0.61 mmol / g. The general procedure for SPPS is substantially as described herein. Preparation 16 is obtained from the soft cleavage of the peptide on the resin described herein using methods known to those skilled in the art. Reconstitution of the concentrated material is carried out with ethanol (5% vol of the combined filtrate) and concentration to dryness. The peptide is ground with stirring in water (40% vol of the combined filtrate). The solid is isolated and dried under vacuum at 40°C to a constant weight to obtain 5.24 g (99%) of Preparation 16 as a white powder.

[0063] Synthesis of preparation 18 Sequence ID 17 In the synthesis, Fmoc-Ala-OH2-chlorotrityl resin is used at a loading of 0.50 mmol / g. The general SPPS procedure is used substantially as described herein, with the following modifications. [Table 7]

[0064] Preparation 18 Soft Cut: A 42.13 g sample of the peptide on the resin intermediate was placed in a flask and treated three times with 10 times the volume (400 mL) of 1% TFA / DCM for 10 minutes each time, followed by washing with DCM. Each treatment was quenched by adding 4.4 mL of pyridine. The resulting solutions were combined and concentrated under vacuum. Reconstitution was carried out with ethanol (25 mL), followed by concentration to dryness to obtain 56.6 g of foamy semi-solid. 400 mL of water was added 10 times to produce a slurry. The slurry was filtered and washed with water. The solid was isolated and dried under vacuum at 40°C to a constant weight to obtain 23.3 g of preparation 18 as a white powder.

[0065] 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-tetraazatripentacontan-53-acid) Preparation 6 (80 g, 92 mmol), DIEA (17.53 mL, 101 mmol), TSTU (30.3 g, 101 mmol), and acetonitrile (1 L) are placed in a container and stirred at 23°C for 17 hours. The solution is concentrated, and the resulting orange residue is then redissolved in EtOAC (1.6 L) and washed with 0.1 M HCl (2 × 1 L). The organic layer is washed with water (2 × 1 L), then dried over MgSO4, filtered, and concentrated under vacuum to yield the orange oil (83 g). A second batch is carried out on the same scale, yielding a total of 123 g of crude oil. The intermediate ester (123 g, 110 g active, 113 mmol) is dissolved in EtOH (700 mL), then Fmoc-lysine (45.9 g, 125 mmol) and DIEA (21.70 mL, 125 mmol) are added, and the reaction mixture is stirred for 17 hours. After the reaction is complete, EtOH is removed under vacuum, leaving an orange oil (201 g). The residue is dissolved in SiO2 (1.1 L) and washed with a 0.1 M HCl solution (3 x 400 mL), then with an aqueous NaHCO3 solution (400 mL). The layers are separated, and the organic layer is washed with a saturated sodium chloride aqueous solution (1 x 400 mL). The organic matter is concentrated to yield an orange oil (approximately 190 g). Acetone (400 mL) is added, and then the resulting suspension is filtered to remove the inorganic matter. The mixture is concentrated and purified by normal-phase chromatography (1.1 kg of silica prepared with 60 / 40 heptane / acetone), and the polar eluent is increased to elute (collecting approximately 3 L of fraction). The fractions representing at least 95% of the HPLC area are combined and concentrated to yield Preparation 17, a dark yellow oil (70 g).

[0066] Synthesis of preparation 19A Sequence ID 18 In the synthesis, Fmoc-Leu-OH2-chlorotrityl resin is used at a loading of 0.65 mmol / g. The general SPPS procedure is used with the following modifications. [Table 8] [Table 9]

[0067] Preparation 19A derived from pseudoproline can be treated with Example 3 in the same manner as preparation 19B, as described herein.

[0068] Synthesis of preparation 19B Sequence ID 19 In the synthesis, Fmoc-Leu-OH2-chlorotrityl resin is used at a loading of 0.65 mmol / g. Preparation 19B is prepared using the SPPS procedure substantially as described herein. [Table 10] [Table 11]

[0069] Preparation 19B Soft Cut: Preparation 19B is prepared using a method known to those skilled in the art, substantially as described herein, by soft cutting of 19B bonded to the resin. See, for example, the method for Preparation 18. The resulting solid is isolated and dried under vacuum at 30-40°C to a constant weight to obtain 2.94 g of the product as a pale yellow powder.

[0070] Synthesis of preparation 20 Sequence ID 20 To the solutions 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), PyBOP (2.60 g, 5.00 mmol) and DIEA (1.75 mL, 10.0 mmol) were added at ambient temperature. The solution was stirred for 18 hours and quenched by adding excess diethylamine (10.0 mL) with water. The quenched solution was stirred for 2 hours and then slowly added to a saturated sodium bicarbonate aqueous solution / water (1:1, 300 mL) at 0°C. The resulting precipitate was stirred for 10 minutes and then collected by filtration. The filtrate was washed sequentially with water (3 x 150 mL) followed by methyl tert-butyl ether (3 x 150 mL). The solid is dried under vacuum at 40°C to obtain preparation 20 as a white solid (5.30 g, 69% yield, C 119 H 169 N 21 O 24 (HRMS calculation predicted value: 2276.2649, actual value: 2276.2652).

[0071] Synthesis of preparation 21 Sequence ID: 21 To the solutions 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), PyBOP (314 mg, 0.30 mmol) and DIEA (0.21 mL, 1.20 mmol) were added at ambient temperature. The solutions were stirred for 18 hours, then quenched with piperidine (0.79 mL, 4.00 mmol). The quenched solutions were stirred for 2 hours, then cooled to 0°C, and quenched with a dilute solution of HCl (50 mL). The resulting slurry was stirred for 10 minutes, and the solid was collected by filtration. The filtrate was washed sequentially with saturated sodium bicarbonate aqueous solution (2 x 50 mL), water (3 x 50 mL), and then methyl tert-butyl ether (3 x 50 mL). The solid was dried under vacuum at 40°C for 18 hours to obtain preparation 21 as a white solid (1.80 g, 106% yield, C 225 H 338 N 34 O 48 (HRMS calculated value: 4284.5053, measured value: 4284.5062).

[0072] Synthesis of preparation 22 Sequence ID: 22 To the solutions of Preparation 21 (214 mg, 0.05 mmol) and Preparation 19B (116 mg, 0.055 mmol) in DMSO / DMF (1:1, 3 mL), PyBOP (57 mg, 0.11 mmol) and DIEA (58 μL, 0.33 mmol) were added at ambient temperature. The solutions were stirred for 18 hours, then quenched with a 1:1 mixture of saturated sodium bicarbonate aqueous solution and water (10 mL). The mixture was stirred for 10 minutes, and the resulting solid was collected. The solid was washed with water (3 x 10 mL), and the solid was dried under vacuum at 40°C to obtain Preparation 22 (285 mg, 89% yield, C). 334 H 512 N 48 O 74 (HRMS calculation predicted value: 6379.7777, actual value: 6379.7730).

[0073] Example 3 Synthesis of Example 3 Sequence ID 1 A solution of TFA (2.3 mL), water (0.1 mL), triisopropylsilane (0.1 mL), and DTT (75 mg) is cooled to 0°C. Preparation 22 (100 mg, 0.015 mmol) is added to the solution, and the reaction mixture is heated to ambient temperature and stirred for 2 hours. The resulting mixture is poured into a pre-cooled (-20°C) solution of methyl tert-butyl ether (25 mL). The resulting precipitate is maintained at -20°C for 15 minutes, the slurry is centrifuged, and washed with methyl tert-butyl ether (2 x 25 mL). The solid is dried under vacuum at 35°C for 18 hours to obtain Example 3 as a white solid (71 mg, 93% yield, C 225 H 348 N 46 O 68 (HRMS calculation predicted value: 4810.5249, actual value: 4810.5036).

[0074] Step 1 Step 1: Prepare a feed solution for preparation 25 (1.05 equivalents) with 5 vol of DMSO / ACN (90:10 vol / vol). Prepare a second feed solution for preparation 26 with 20 vol of DMSO / ACN (90:10 vol / vol). A third feed solution is prepared from 1.5 equivalents of PyOxim in 3 vol of ACN. A fourth stream (4 equivalents) of DIEA in ACN is prepared. The first three streams are pumped to a mixer, where DIEA is mixed at the mixer outlet, and the mixture is sent to a 20°C bath via another mixer and a plugged flow reactor, where it remains for 2 hours. At the reactor outlet, acetic acid can be added to consume the remaining PyOxim (1-cyano-2-ethoxy-2-oxoethylideneaminooxy-tris-pyrrolidino-phosphonium hexafluorophosphate). After more than 2 hours, the stock solution of diethylamine (10 equivalents) is added and mixed with a mixer. This flow proceeds to the second plugged flow reactor and is kept in a 20°C constant temperature bath for 1 hour. The product solution of preparation 27 is collected and sent to nanofiltration using a 70 / 30 DMSO / ACN solution to remove 10-20 diavolutes of reagent.

[0075] The feed solution for Preparation 25 (2.40 kg, 96.7 wt%, 2.274 mol) is 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), and a solution of Preparation 25 (114.3 mg / mL, 0.112 M) is prepared with DMSO:ACN at a ratio of 90:10 vol / vol. The second feed solution for Preparation 26 (SEQ ID NO: 26, 2.79 kg, 98.6 wt%, 1.836 mol) is prepared by dissolving the solid in DMSO (54.8 kg, 49.8 L) and diluting the solution with ACN (4.3 kg, 5.47 L), and a solution of Preparation 26 (45.5 mg / mL, 0.030 M) is prepared with DMSO:ACN at a ratio of 90:10 vol / vol. The third feed solution is prepared with PyOxim (4.5 kg, 8.53 mol) in ACN (47.33 kg, 60.22 L) to produce a 0.132 M solution. DIEA is added in stock form. The streams of solution 25 (14.91 L, 1.704 kg, 1.670 mol, 0.95 equivalents, 5.9 g / min), solution 26 (58.46 L, 2.660 kg, 1.750 mol, 1.00 equivalent, 22.5 g / min), and PyOxim (1.4 equivalents, 5.4 g / min) are mixed with stock DIEA (4.0 equivalents, 0.446 mL / min) at 20°C and pumped into the mixer. The mixture is pumped to a 20°C constant temperature bath via another mixer and a plugged flow reactor, and after a 3-hour retention period, collection over 42.9 hours yields 88.6 kg of product solution.

[0076] Nanofiltration is a membrane-based filtration process used to separate chemical species based on differences in size and molecular weight. The product solution of Preparation 27 contains reagents (such as diethylamine, PyOxim, and DIEA) and unwanted by-products (e.g., dibenzofluben) that need to be removed before proceeding to the next step. Nanofiltration is applied to remove undesirable species (molecular weight less than 500 Da).

[0077] The product solution of Preparation 27 is filled into an NF feed tank and pumped through a recirculation loop via a heat exchanger and a nanofiltration unit containing a suitable membrane (ceramic or polymer) to induce the desired separation. Unwanted species are removed from the permeate and collected separately or discarded. To maintain a constant volume in the NF tank, a fresh solvent, i.e., 70:30 vol / vol DMSO / ACN, is continuously pumped in to match the rate of permeation being drawn. The product solution of Preparation 27 is purified by nanofiltration and transported directly to step 2.

[0078] A solution of Fmoc-protected preparation 27 in DMSO / ACN (88.6 kg) and diethylamine (1.34 kg) is added to the reactor. The mixture is stirred at 20°C for 2 hours to yield preparation 27 (87.6 L, 38.45 mg / mL, 3.37 kg, 1.48 mol). The product solution of preparation 27 is filled into a nanofiltration feed tank and then pumped through a recirculation loop via a heat exchanger and a nanofiltration unit containing a suitable membrane (ceramic or polymer) to induce the desired separation. Unwanted species are removed on the permeate side and collected separately or discarded. This operation is continued until all unwanted impurities are sufficiently removed. To maintain a constant volume in the nanofiltration tank, new 70:30 vol / vol of DMSO / ACN is continuously pumped in to match the rate of permeation being drawn. This yielded preparation 27 with DMSO / ACN (72.4 L, 40.8 mg / mL, 2.95 kg, 1.30 mol, 78.1% yield overall after coupling, Fmoc removal, and nanofiltration).

[0079] Step 1 Example - Analysis Results. HPLC confirms the conversion of preparations 25 and 26 to preparation 27. The analytical method uses a phenylhexyl stationary phase column (2.1 mm inner diameter x 150 mm inner diameter x 1.7 micron particle size) at 65°C, with a 2-98% B gradient of 0.1% TFA in water and acetonitrile for 12 minutes. UV detection at 214 nm is used for this material.

[0080] Table A.1 shows high-resolution mass spectrometry data collected for the product of the coupling reaction in Step 1 (Fmoc protected preparation 27) and the product of the deprotection reaction in Step 1 (preparation 27). Mass precision is the metric used to confirm the agreement between the species being measured and the species being predicted. [Table 12] Table A.1 Confirmation of Fmoc-protected preparation 27 measured and preparation 27 by mass accuracy calculated using high-resolution mass spectrometry data.

[0081] Step 2 [ka]

[0082] Schematic Figure A.2 Synthesis of preparation 29 (sequence number 29) from fragment preparation 27 (sequence number 27) and preparation 28 (sequence number 28). [ka]

[0083] Step 2: Prepare a feed solution of Preparation 28 (1.15 equivalents) with 10 vols of DMSO / ACN (90:10 vol / vol). Prepare a second feed solution with 2 equivalents of PyOxim in 1 vol of ACN. Prepare a third stream of DIEA (3 equivalents) in ACN (5 wt% solution). The streams of Preparation 27 solution from Step 1, Preparation 28, and PyOxim are pumped into a mixer, where they are mixed with DIEA at the mixer outlet. The mixture is pumped into a 20°C bath via another mixer and a plugged flow reactor, where it remains for 2 hours. At the reactor outlet, acetic acid can be added to consume any remaining PyOxim. After 2 hours, 10 equivalents of stock diethylamine are added and mixed in a mixer. This stream reaches a second plugged flow reactor and remains in a 20°C bath for 1 hour. The product solution of preparation 29 is collected, and the DMF solution is sent to nanofiltration as a diafiltrant to remove 10-20 diavolutes of reagent.

[0084] The feed solution for Preparation 28 (4.68 kg, 98.9 wt%, 2.058 mol) is 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), and a solution of Preparation 28 (94.7 mg / mL, 0.0421 M) is prepared with DMSO:ACN in a 90:10 vol / vol ratio. The second feed solution is prepared with PyOxim (3.0 kg, 5.69 mol) in ACN (11.81 kg, 15.03 L) to produce a 0.327 M solution. DIEA is added stock. The solutions of Preparation 27 from Step 1 (73.86 L, 41.2 mg / mL, 3.04 kg, 1.336 mol, 0.0181 M, 1.0 equivalent, 29.9 g / min), Preparation 28 (1.3 equivalents, 17.7 g / min), and PyOxim (2.1 equivalents, 2.9 g / min) are pumped into a mixer, where the flows are adjusted to 20°C and mixed with the stock solution of DIEA (4.0 equivalents, 0.374 mL / min) at 20°C. The mixture is pumped into a 20°C bath via another mixer and a plugged flow reactor, and after a 3-hour retention period, collection over 43.2 hours yields 129.35 kg of Preparation 29 product solution.

[0085] In effect, the nanofiltration process described above uses the product solution of preparation 29 instead of the product solution of preparation 27.

[0086] The nanofiltration process using a solution of Fmoc-protected preparation 29 in DMSO / ACN is carried out substantially as described herein. Fmoc-protected preparation 29 (129.35 kg) and diethylamine (2.0 kg) are placed in a reactor for nanofiltration. The nanofiltration process yields preparation 29 in DMF (98.85 L, 42.24 mg / mL, 4.18 kg, 0.974 mol, with a yield of 73.1% overall after coupling, de-Fmoc removal, and nanofiltration).

[0087] HPLC confirms the synthesis of preparation 29 from preparation 28 and preparation 27. The analytical method uses a C4 stationary phase column (2.1 mm inner diameter x 150 mm inner diameter x 1.7 micron particle size) at 65°C, with a B gradient of 25–98% in water and 0.1% TFA in acetonitrile for 12 minutes. UV detection at 214 nm is used for this material.

[0088] Table A.3 shows high-resolution mass spectrometry data collected for the products of the coupling reaction in Step 2 (Fmoc protected preparation 29) and the deprotection reaction in Step 2 (preparation 29). The mass precision allows for the identification of the product of the measured species and the monoisotopic mass of the neutral species. [Table 13] Table A.3 Measured Fmoc-protected preparation 29 and confirmation of preparation 29 by mass accuracy calculated using high-resolution mass spectrometry data.

[0089] Step 3 Schematic Figure A.3 Synthesis of preparation 31 (sequence number 31) from fragment preparation 30 (sequence number 30) and preparation 29 (sequence number 29). [ka]

[0090] Step 3 Batch Process Description: Preparation 29 (2.249 g, 46.5 mg / g, 104.6 b mg, 0.0244 mmol) and Preparation 30 (71.5 mg, 90.6 area%, 0.0306 mmol) are nanofiltered into DMF (0.3068 g, 0.325 mL) at -5°C. A 5.0 wt% DIEA solution in DMF (114.0 mg, 5.70 mg of DIEA, 0.0441 mmol, 1.8 equivalents) and a 10.1 wt% solution in DMF (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxidehexafluorophosphate) are added. Add phatate (HATU) (154.9 mg, 15.59 mg of HATU, 0.0410 mmol, 1.7 equivalents). Stir the solution at -5°C for 4 hours, then quench by adding 5 wt% sodium bicarbonate aqueous solution (5.295 g, 4.8 mL) over 15 minutes at ambient temperature. Stir the resulting slurry at 0°C for 15 minutes, and collect the resulting solid by filtration. Wash the solid with water (4 x 2 mL), then wash with MTBE (4 x 2 mL). Dry the viscous solid under vacuum at 35°C to obtain preparation 31 (219.7 mg, 47.6 area%, 0.0164 mmol, 67.1% yield).

[0091] A feed solution of preparation 30 is prepared in 10 volumes of DMF. A second feed solution is prepared with HATU (1.8 equivalents) in a 10 wt% solution containing ACN. A third flow of DIEA (2.5 equivalents) is prepared in DMF (5 wt% solution). The flows of preparation 29 from step 2, preparation 30, and DIEA are pumped to a mixer, where the flows are cooled and mixed with the cooled HATU solution at the mixer outlet. The mixture is pumped through another mixer and a plugged flow reactor to a -5°C constant temperature bath, where it is collected after 3 hours of retention. A 19 wt% salt load is prepared in a brine / bicarbonate solution (17 wt% aqueous sodium chloride solution, 0.5 wt% sodium bicarbonate solution). The product solution of preparation 31 in DMF is then pumped together with the salt solution to a mixed product mixed suspension reactor (MSMPR) to form a precipitate. The tau of the mixed product suspension reactor is 1 hour. A second MSMPR is performed at low temperature for 1 hour tau, and this slurry is intermittently packed into a filter. The slurry is washed with water and dried under vacuum at 35°C.

[0092] A feed solution of preparation 30 (9.42 kg, 83.6 wt%, 3.72 mol) is prepared in DMF (54.12 kg) to create a 0.0563 M feed. A second feed solution is prepared in HATU (1.15 kg, 3.02 mol) in ACN (10.4 kg) to create a 0.215 M feed. DIEA is added stock. The streams of preparation 29 (98.85 L, 42.24 mg / mL, 4.18 kg, 0.975 mol, 0.0099 M, 1.0 equivalent, 40.2 g / min), preparation 30 (1.3 equivalents, 9.2 g / min), and DIEA (2.1 equivalents, 0.152 mL / min) from step 2 are pumped into the mixer. At the mixer outlet, the flow is cooled to 0°C and mixed with HATU solution (2.0 equivalents, 3.2 g / min) cooled to 0°C. The mixture is pumped through another mixer and a plugged flow reactor to a constant temperature bath at 0°C, where it is collected over 42 hours after a 4-hour retention period, yielding 131.8 kg of product solution. The product solution is allowed to precipitate in two sections. 17 wt% NaCl aqueous solution / 0.5 wt% NaHCO3 aqueous solution (29 kg) is mixed with DMF (13.2 kg) in an inactivating reactor and cooled to below 20°C. Then, the product solution in DMF (66.6 kg) is co-added to the reactor with 17 wt% NaCl aqueous solution / 0.5 wt% NaHCO3 aqueous solution (34.4 kg) over 1 hour, maintained at 20°C, yielding product precipitation and preparation 31. The slurry is cooled to 5°C over 1 hour, then water is added in two batches (total 32.2 kg). Before filtering the slurry, the 5°C slurry is stirred for 0.5 hours. The wet cake is re-slurried with water (63.9 kg) for 0.5 hours and filtered. The second section of the product solution is precipitated in the same manner. The solution of 17 wt% NaCl aqueous solution / 0.5 wt% NaHCO3 aqueous solution (29 kg) and DMF (13.55 kg) is mixed in the reactor and cooled to below 20°C. The second section of the product solution in DMF (65.2 kg) is co-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 precipitate the product and yield preparation 31. The slurry is cooled to 5°C over 1.25 hours, then water is added in two batches (total 32.1 kg).The slurry is stirred for 0.5 hours and filtered over the first wet cake. The combined wet cake is re-slurried twice with water (64 kg each) for 0.5 hours each time and filtered. This is followed by two displacement washes with water (64 kg each). N2 is blown into the combined washed wet cake and then dried under vacuum at 38°C until the KF is less than 4 wt%, yielding Preparation 31 (10.34 kg, estimated potency 60%, 0.972 mol, estimated yield 100%).

[0093] Example 3 Chilzepatide (SEQ ID NO: 1) [ka]

[0094] A solution of TFA (2.3 mL), water (0.1 mL), triisopropylsilane (TIPS, 0.1 mL), and dithiothreitol (DTT, 75 mg) is cooled to 0°C. Preparation 31 (100 mg, 0.015 mmol) is added to this solution, and the reaction mixture is heated to ambient temperature and stirred for 2 hours. The resulting mixture is poured into a pre-cooled (-20°C) MTBE solution (25 mL). The resulting precipitate is maintained at -20°C for 15 minutes, the slurry is centrifuged, and washed with MTBE (2 x 25 mL). The solid is dried under vacuum at 35°C for 18 hours to obtain Example 3 as a white solid (71 mg, 93% yield, C 225 H 348 N 46 O 68 (HRMS calculation predicted value: 4810.5249, actual value: 4810.5036).

[0095] In a deactivating reactor at 15°C, add DCM (27.3 kg, 20.6 L), water (4.1 kg), preparation 31 (10.34 kg, estimated potency 60%, 0.972 mol), and DTT (3.10 kg). In another deactivating reactor, add TFA (154.1 kg, 103.4 L) and TIPS (3.2 kg, 4.2 L). Add the TFA / TIPS solution to the slurry of preparation 31, DCM, water, and DTT within 0.25 hours to form a colorless solution, which is then heated and held at 20°C for 3 hours. After 3 hours at 20°C, cool the reactor to -10°C. Add MTBE (382.4 kg, 516.8 L) to another reactor and cool to -20°C. A portion of this cold MTBE (91.8 kg, 124.1 L) is added to the cold reaction solution over 2 hours while maintaining a temperature of -5°C to -18°C. The remaining cold MTBE (294.3 kg, 397.7 L) is added over 1.5 hours while maintaining a temperature of -5°C to -18°C to yield the precipitate of Example 3. The slurry is adjusted to 0°C and held for more than 0.5 hours, then filtered into three sections. The wet cakes are combined and re-slurried twice with MTBE (114.7 kg, 155 L), filtered, and then subjected to a final MTBE substitution wash (114.7 kg, 155 L). The wet cakes are dried at 28°C until less than 4.5 wt% MTBE is measured. This yields Example 3 (7.77 kg, 46.8 wt%, 0.755 mol, 77.7% yield).

[0096] Example 4A (Linear SPPS) Chilzepatide (SEQ ID NO: 1) [ka] [ka]

[0097] Fmoc Sieber resin (17 kg, 0.76 mmol / g) is packed into the reactor. The resin is swollen with DMF and stirred for 2 hours, then filtered to remove the DMF. The resin is then washed twice with DMF. The Fmoc-protected resin is then deprotected using a 20% PIP / NMP treatment. Sampling to confirm Fmoc removal is performed after the final PIP / NMP treatment, and UV analysis confirms over 99% Fmoc removal (IPC target residual Fmoc less than 1%). After the final 20% w / w PIP / NMP treatment, the resin bed is washed multiple times with DMF. The peptide skeleton is then constructed using the following general conditions for coupling and deprotection of each amino acid. [Table 14]

[0098] Fmoc deprotection: The resin in the peptide reactor is treated by filling it with a 20% v / v PIP / NMP solution three or four times. After stirring on the resin for 30 minutes after each treatment, it is filtered to complete the removal of the Fmoc protecting group. After the final 20% v / v PIP / NMP treatment, the resin bed is washed at least six times with DMF filled to the pre-specified volume.

[0099] Amino acid activation: A pre-prepared solution of 12% w / w Oxyma Pure / NMP is packed into the reactor. The selected Fmoc amino acid is then added. The mixture is stirred at 20±5°C until the Fmoc amino acid is completely dissolved. To ensure control of the slight exothermic activation reaction, the Fmoc-AA / Oxyma Pure / NMP solution is then cooled to 15±3°C before activation, and the resulting solution temperature is maintained within the specified range of 20±5°C. The amino acid solution is then activated by DIC addition. The activated ester solution is then stirred for 20-30 minutes before transferring the solution to the reactor containing the peptide on the resin intermediate.

[0100] Coupling: Once the pre-activation step is complete, the activated ester solution is transferred to a 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 for intermittent coupling (IPC). Sampling is repeated at specific intervals as needed until a satisfactory IPC result is obtained. Re-coupling is performed as needed. Once coupling is complete, the contents of the peptide reactor solution are filtered, and the peptide on the resin intermediate is washed several times with DMF to prepare for the next coupling.

[0101] Coupling of Ile(12) to Aib(13): The coupling of Fmoc-Ile(12) to Aib(13) is performed using a symmetric anhydride approach with 6 equivalents of Fmoc-AA and 3 equivalents of DIC. To ensure the formation of activated symmetric anhydride species, the activation time of this sequence is extended by 40–60 minutes. To achieve reaction completion (less than 1% non-coupling) as determined by HPLC analysis, the coupling stirring time needs to be extended (18 hours).

[0102] Lys(20)ivDe deprotection (Preparation 23): Selective deprotection of the 39-amino acid Lys(20)ivDde group, which is completely protected by the resin Boc-Tyr(1)-Ser(39) peptide backbone, is performed. Deprotection is achieved by stirring at ambient temperature for 4 hours using 8% w / w hydrazine hydrate in DMF solution. The deprotection reaction is monitored by HPLC, with the target IPC limit being less than 1% of the remaining Lys(ivDde) ​​component after deprotection. The obtained peptide fragment (Preparation 23) is repeatedly washed with DMF (8 times) to completely remove residual hydrazine. The fully constructed Preparation 23 fragment is washed 4 times with IPA and dried at 40°C or below until the LOD is less than 1%. Preparation 23 is packaged and refrigerated (-20°C) before coupling with Preparation 6.

[0103] Coupling of Preparation 6 to Preparation 23: Solids of Preparation 6 (1.5 equivalents) and PyBOP (1.5 equivalents) are packed into a reactor, followed by the addition of DMF, and the mixture is stirred until dissolution occurs. Collidine is then packed in to initiate the formation of the active ester species. The activated ester solution is stirred for 60 minutes, and then transferred to a reactor containing the Preparation 23 intermediate. The reaction slurry is stirred at 25°C for 18 hours. The slurry is sampled for coupling completion (IPC), and sampling is repeated at specific intervals as needed to achieve an acceptable IPC result (Preparation 23 less than 1%). Once coupling is complete, the contents of the solution are filtered and discarded. The fully constructed Preparation 24 intermediate is washed multiple times with DMF, and then with IPA. Preparation 24 is dried at 40°C or below until an LOD of less than 1% is achieved. Preparation 24 is packaged and refrigerated (-20°C) before cutting it from the resin.

[0104] Resin cleavage and rough separation in Example 4A: Prepare a cleavage cocktail consisting of trifluoroacetic acid (TFA), triisopropylsilane (TIPS), dithiotheritol (DTT), DCM, and water. Cool the cleavage cocktail to 15±5°C. The reagent packing is shown in the table below. [Table 15]

[0105] Prepared mixture 24 is packed into the reactor, followed by the cutting cocktail. The reaction mixture is stirred at 23°C for 3 hours. The mixture is filtered, and the spent resin is washed with DCM. The DCM washing filtrate is mixed with the bulk deprotection solution, and the contents are cooled to below -10°C. The MTBE is cooled to below -13°C, and then the cold MTBE is supplied to two parts of the cold filtrate. The MTBE supply rate is controlled to maintain the internal temperature of the crude solution below 5°C. The first MTBE packing constitutes approximately 45% of the total MTBE packing volume. A soft precipitate forms near the end of the MTBE addition, but it redissolves easily in the solution. The precipitate solution is then recooled to an internal temperature of -15±5°C. The second MTBE addition is supplied at a rate of approximately 5 to 10 times that of the first MTBE supply rate, and constitutes approximately 55% of the total MTBE packing volume. During the addition, the internal temperature of the precipitate slurry is maintained below 0°C. The resulting slurry is aged at -8±3°C for a minimum of 6 hours, then warmed to 0±3°C and aged for another 2 hours before isolation.

[0106] 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 until the IPC target LOD value was 1% or less. The crude product of Example 4A was packaged and stored. The crude intermediate was stored refrigerated (-20°C) until purified. Overall, 45.39 kg of crude Example 4A was produced with a purity of 45 wt% and 64% HPLC area percentage. Content yield based on Sieber resin = 47%.

[0107] 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

[0108] Reverse-phase purification 1 (RP1): Dissolve the crude product from Example 4A in 90 wt% MPA and 10 wt% MPB. Stir the solution for at least 7 hours to complete the decarboxylation of tryptophan. Filter the matured crude solution and pack it into a pre-equilibrated Kromasil 100-10-C8 packed column. Wash the column with a mixture of A buffer (90% water, 0.1% TFA, 10% ACN) and B buffer (10% water, 0.1% TFA, 90% ACN) until 30% ACN per two column volumes, and prepare for elution by increasing the ACN mixture concentration from 30% to 35% per column volume. Elute tilzepatide from the column by increasing the ACN by 1.5% per column volume until elution is complete. Fractionate the eluate and assay its purity using RP-HPLC. The column is regenerated by increasing the ACN from 47% to 65% of the first column volume and continuously flowing 65% of the third column volume of ACN. Before the next injection sequence, the column is re-equilibriumated using 30% of the second column volume of ACN.

[0109] Pool the fractions eligible for mainstream inclusion. After all primary injections are complete, fractions that do not meet the purity criteria but have a purity above 50% can be mixed for re-injection. The re-injection fraction is divided into front and back fractions, diluted with buffer A, and stored under refrigeration. The re-injection fraction is processed and pooled using the primary injection criteria, but only the major peak fraction is processed forward, and no further recycling is performed. Once the mainstream pooling is complete, the concentration and purity of the intermediate are assayed. Before RP2 processing, the material is diluted and the pH is adjusted to pH 8. The RP1 process yields 37 kg of crude product and 14,277 g of inclusion product with an average pool purity of 90.9%.

[0110] Reverse-phase purification 2: Pack the RP1 solution from Example 4A into a pre-equilibrated Kromasil 100-10-C8 packed column. Wash the column 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) to obtain a 2-column volume of 20% ACN solution. Elute tilzepatide from the column by increasing the ACN by 3.5% per column until elution is complete. Fractionate the eluate and assay its purity using RP-HPLC. After elution, regenerate the column by increasing the ACN to 80% per column volume and continuing to run 3-column volume of 80% ACN. Re-equilibrate the column with 2-column volume of 20% ACN before the next injection sequence.

[0111] Pool the fractions that qualify for mainstream inclusion. After all primary injections are complete, fractions that do not meet the purity criteria but have a purity above 60% can be mixed for re-injection. The re-injection fractions are separated into front and back pools, diluted with buffer C, and stored under refrigeration. The re-injection fractions are processed and pooled using the primary injection criteria, but only the major peak fractions are processed forward, and no further recycling is performed. Once the mainstream pooling is complete, the concentration and purity of the intermediates are assayed. The pH of the material can be adjusted to 8.0 in preparation for the TFF step. In the RP2 process starting with 14.2 kg, 10.9 kg of contained product is obtained in a yield of 76.7%.

[0112] Ion exchange chromatography (IEX): The RP2 solution of Example 4A was filtered and packed onto an Amberchrom CG-300M column. Two fractions were eluted using mobile phase E (10% ammonium acetate aqueous solution, 5% IPA, pH 8) and mobile phase F (isopropanol). The fractions were analyzed for peptide content, and those with less than 3 mg / mL were discarded. The pooled fraction of the concentrate was stored at 20°C before precipitation. The IEX process was started with 10.9 kg of RP2 and yielded 14.3 kg of Example 4A material-containing product with a pool purity of 97.8%.

[0113] Precipitation: Filter the IEX solution (333 kg) from Example 4A, then fill with isopropanol (850 L) to reduce the water content to less than 10% w / w. Cool the diluted solution to 0 ± 3°C in preparation for MTBE filling and precipitation. Cool MTBE (2304 L, 1708 kg) to 0 ± 3°C. Feed the cold MTBE into the IEX solution at a rate of approximately 0.69 kg / min until the first approximately 37% of the MTBE is filled. Then increase the feed rate to an average of approximately 2.3 kg / min to complete the remaining approximately 63% of the MTBE filling. Maintain the temperature below 5°C during feeding. Cold filter the resulting precipitated slurry (below -10°C), then wash the filter cake with MTBE. Dry the filter cake to a LOD of less than 2%.

[0114] Humidification 4A: Example 4A is humidified by passing moist nitrogen through a filter dryer. The humidity of the gas flow exiting the filter outlet is monitored every 60 minutes. Humidification is continued until less than 0.5% MTBE and less than 0.2% IPA remain in the wet cake. After the completion of the humidification process, the nitrogen flow is switched so that dry nitrogen flows through the pure product cake of Example 4A. The material is sampled for water and residual solvent against specific IPC targets, and drying with dry nitrogen is continued until the desired target moisture content of 5-7% w / w is achieved. A total of 12.9 kg of Example 4A is isolated with a purity of over 95% peptide content. Overall yield based on Sieber resin filling = 31%.

[0115] Example 4B (Linear SPPS) Chilzepatide (SEQ ID NO: 1) [ka] [ka]

[0116] Preparation 23 The process for producing Preparation 23 is substantially the same as described in Example 4A, except that NMP is replaced entirely with DMF for all coupling and deprotection. Furthermore, the stoichiometric amounts of amino acids:Oxyma:DIC are reduced to 2.5:2.5:2.7 molar equivalents based on the Sieber resin. The only exception related to the use of DMF is the coupling of Ile12 to Aib13, where NMP is retained. This example shows the process up to 92.2 kg of Preparation 23 peptide on a resin intermediate of 17.6 kg of Sieber resin.

[0117] Preparation 24 The process substantially described in Example 4A involves preparing 92.1 kg of preparation 23, and further processing 97.3 kg of preparation 24 peptide with a resin intermediate. Preparation 24 is packaged and refrigerated (-20°C) before being cut from the resin.

[0118] Resin cutting and crude isolation of Example 4B: Using conditions substantially described in Example 4A, two batches were prepared in 32 kg scale preparation 24, delivering 24.4 kg of Example 4B with 69.5% HPLC purity and 52.6% yield, and 21.3 kg of Example 4B with 88.3% HPLC purity and 45.2% yield. The crude intermediate was stored cold (-20°C) until purified.

[0119] Purification of Example 4B: Crude Dissolution: Crude tilzepatide Example 4B is packed into a dissolution vessel and dissolved in a 1:1 acetonitrile:aqueous solution to a final solution concentration of 25 g solid / L. The pH of the resulting solution is adjusted to 8.5-9.5 with ammonium hydroxide to initiate the conversion of the depsipeptide isomer (10-15%) to tilzepatide Example 4B. The pH-adjusted mixture is stirred for at least 1 hour to allow the depsipeptide conversion to occur. Trifluoroacetic acid is then added to adjust the pH to 1.5-2.5 and diluted to a 30% acetonitrile content in preparation for chromatography. In total, the crude solution is stirred for at least 7 hours to convert the Trp CO2 salt to tilzepatide Example 4B.

[0120] Conversion of tilzepatide (TZP) to depsipeptides: [ka]

[0121] Conversion of depsipeptides to APIs: [ka]

[0122] Reverse-phase purification 1 (RP1): The depsipeptide can be converted to API using an RP1 process substantially as presented in Example 4A. The RP1 purification process is substantially the same as that described in Example 4A. However, the crude dissolution step described above enhances the function of the RP1 chromatography step. This allows for a higher g Tirzepatide per L resin load and reduces the number of injections required to purify the crude Tirzepatide under the conditions described in Example 4. In this example, 23.7 kg of content-corrected crude Example 4B yields 25.4 kg of Example 4B (107%) after RP1. With a total solution volume of 2910 L @ 8.72 g / L, once all pool fractions have been collected, the mixture is stirred, sampled, and held before reverse-phase purification 2 (RP2).

[0123] Reverse-phase purification 2 (RP2): This is substantially the same purification process as described in Example 4A and is used in reverse-phase purification 2 (RP2) using methods known to those skilled in the art. In this example, 15.2 kg of Example 4B from RP1 is purified to 13.8 kg of Example 4B with a purity of approximately 98% after RP2. Total solution volume = 808 L @ 17.0 g / L. The mixture is stored before tangent flow filtration.

[0124] Tangent flow filtration (TFF): The TFF membrane is set up and washed with water. Ammonium acetate buffer is prepared using low-endotoxin purified water, acetic acid, and ammonium hydroxide. Isopropanol is then added to supply 100 mM NH4OAc pH8.0:IPA buffer in a 5:95 ratio. The 17 g / L RP2 solution from Example 4B is concentrated to approximately 125 g / L through the TFF. The RP2 solution is recirculated to allow the solvent to permeate the membrane while retaining the peptide solution on the retaining liquid side of the membrane in the solution. After concentration, the diafiltration buffer is supplied to the retaining liquid tank while the permeate is continuously collected. Buffer exchange is continued until the desired solvent composition and peptide concentration are achieved. The solution is emptied from the system, and the resulting polarization layer is washed from the membrane and pooled together with the peptide concentrate. Two sections of the RP2 solution (403.9 L @ 17 g / L, 9.87 kg API) are processed through the TFF.

[0125] Simultaneous Feed Precipitation: Combine the TFF sections (138.2 kg, 78.3 g / L), measure the KF (8.9%), and confirm that the water content is less than 10%. Place MTBE (243 kg) in a separate container and cool to 0°C. Add IPA (48 kg), water (6 kg), and MTBE (100 kg) to the precipitation vessel and cool the solution to 0°C. The TFF and MTBE process flows are simultaneously fed into the precipitation vessel at rates of 1.6–1.8 kg / min and 2.9–3.1 kg / min, respectively. Allow the resulting slurry to mature at 0°C for a further 0.7 hours, then warm to 15°C. After maturing the slurry at 15°C for 1 hour, add MTBE (118 kg). After maturing the slurry at 15°C for 1 hour, cool to 2.5°C. Cold filter the slurry and wash the filter cake with MTBE (573 kg). Dry the filter cake to less than 2% LOD.

[0126] Humidification: Humidification, substantially as shown in Example 4A, is applied to the material of Example 4B using methods known to those skilled in the art. A total of 14.5 kg of Example 4B (SEQ ID NO: 1) is isolated with HPLC purity exceeding 97.7% and peptide content of 88.4%. Overall yield based on Sieber resin filling = 46%.

[0127] Example 5 Serial synthesis of preparation 31 using convergent chemistry in a flow The synthesis of preparation 31 from peptide fragments is carried out using both a batch approach to chemistry and the sequential addition of fragments in a tubular flow reactor. The generalized approach to synthesis involves coupling the two fragments by blending the two solutions with a coupling agent into a tubular reactor, followed by the addition of a base and an extended residence time in the tubular reactor to remove the FMOC protecting group. This prepares a coupled species for the addition of the next fragment. Excess reagents, bases, and solvents are removed during the sequential coupling reaction using nanofilters with membranes sized to retain the peptides and penetrate low molecular weight impurities. Diafiltration is used to completely remove low molecular weight impurities before the subsequent coupling step. An explanation and analytical results from examples of these transformations are given below.

[0128] HPLC is used to confirm the synthesis of preparation 31 from preparations 29 and 30. The analytical method uses a C4 stationary phase column (2.1 mm inner diameter x 150 mm inner diameter x 1.7 micron particle size) at 65°C, with a 60–98% B gradient of 0.1% TFA in water and acetonitrile for 12 minutes. UV detection at 214 nm is used for this material.

[0129] Table A.5 shows high-resolution mass spectrometry data collected for the product (preparation 31) of the step 3 coupling reaction prepared in the flow. The target species can be identified by mass precision. [Table 16] Table A.5 Confirmation of the measured preparation 31 by mass accuracy calculated using high-resolution mass spectrometry data.

[0130] Native chemical ligation is a useful process for preparing full-length peptides containing cysteine ​​or alanine in their sequence. This process utilizes the chemoselective reaction of two unprotected peptide segments to generate a transient thioester-bonded intermediate. The thioester-bonded intermediate is then rearranged to provide a full-length ligation product with a native peptide bond at the ligation site. Those skilled in the art will understand that the technique of native chemical ligation can be useful in the chemical synthesis of full-length peptides containing cysteine ​​or alanine.

[0131] Example 6 Native chemical ligation process Sequence ID 1 [ka]

[0132] Synthesis of Fmoc-hydrazine-CTC resin (Preparation 32) Swell 2-CTC resin (10.7 g, 17.7 mmol) in 100 mL of DCM at 0°C for 20 minutes. Dissolve 9-fluorenyl methyl carbazate (15.6 g, 61.4 mmol, 3.5 equivalents) in 210 mL of 2:1 DMF:DCM. Add DIEA (31 mL, 178 mmol, 10.1 equivalents) to the 9-fluorenyl methyl carbazate solution. Then slowly add this solution to the resin at 0°C. Stir at 0°C for about 1 hour, then raise to room temperature. Stir the reaction mixture at room temperature for 16 hours. Then add methanol (10 mL) to quench the remaining 2-CTC resin and stir for 15 minutes. The resin is rinsed with 200 mL of DMF, followed by DMF (2 x 100 mL), water (3 x 100 mL), DMF (3 x 100 mL), methanol (3 x 100 mL), and finally DCM (3 x 100 mL). The resin is dried in a vacuum oven at 27°C for 16 hours. The resin load is measured to 0.74 mmol / g by quantitative NMR. [ka]

[0133] Synthesis of peptide hydrazide (17-mer) (Preparation 33) Sequence ID 32 Hydrazine-CTC resin (1.01 g, loading value: 0.65 mmol / g) is placed in a 40 mL reaction vessel and swelled on a peptide synthesizer with 3 x 4 mL of DCM (30 seconds each), then swelled with 2 x 10 mL of DMF (20 minutes each). Fmoc-Ile-OH (0.919 g, 2.60 mmol, 4 equivalents) and HBTU (0.99 g, 2.61 mmol, 4 equivalents) are dissolved in 7 mL of DMF. DIPEA (0.91 mL, 5.22 mmol, 8 equivalents) is added to the amino acid solution and the volume is increased to 10 mL 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 x 10 mL of DMF and 5 x 10 mL of DCM, and dried for 12 hours. The load of the obtained resin was measured to be 0.54 mmol / g by quantitative NMR. 0.91 g of this resin was used for the synthesis of preparation 33 (SEQ ID NO: 32).

[0134] Deprotection: 4 x 9 mL of 20% v / v piperidine in DMF, 30 minutes each.

[0135] Coupling: 3 equivalents of amino acids, 3 equivalents of OXYMA, and 3.3 equivalents of DIC are used for amino acid coupling. After each coupling and deprotection iteration, the resin is washed with 5 × 9 mL of DMF for 1 minute with N2 mixture. After peptide hydrazide synthesis, the resin is washed with DCM with N2 mixture. The resin is dried on the synthesizer.

[0136] Deprotection and cleavage: Add a 25 mL cleavage cocktail prepared with 5% w / v dithiothreitol (DTT), 2.5% v / v water, 2.5% v / v triisopropylsilane (TIPS), and 90% trifluoroacetic acid (TFA) to the dried resin (2.37 g) and mix in a rotary mixer for 3 hours. Filter the resin and wash with 2 x 2.5 mL of TFA. Pour the filtrate into 175 mL of cold MTBE and allow the peptide to precipitate immediately. Wash the filtration flask with 2 x 2.0 mL of TFA and pour into cold MTBE. After cooling to -20°C for 30 minutes, centrifuge. Then, wash the peptide precipitate twice with 150 mL of MTBE and centrifuge. Dry the peptide precipitate in a vacuum oven at 27°C for 16 hours. Obtain a 1.25 g sample of crude preparation 33 after drying [predicted value (mass + 2H)]. + ) / 2 = 968.4883, observed value (mass + 2H + ) / 2 = 968.4879]. [ka]

[0137] Approximately 0.62 mmol of preparation 34 is synthesized on Sieber amide resin using a standard SPPS protocol. Fmoc-Lys(ivDde)-OH is used for orthogonal deprotection and lysine acylation.

[0138] Deprotection of ivDde: Dilute hydrazine monohydrate (64% w / w) (1.98 g, 25.3 mmol) with DMF to 24.4 g, and add 20 g to the resin. Stir the slurry with a nitrogen stream. Wash with 5 x 9 mL of DMF after about 2 hours. Repeat once more.

[0139] Dissolve 2-[2-[2-[[2-[2-[2-[[(4S)-5-tert-butoxy-4-[(20-tert-butoxy-20-oxo-icosanoyl)amino]-5-oxo-pentanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetic acid (1094.4 mg, 1.252 mmol, 2 equivalents) in 10 mL of anhydrous DMF. Add TNTU (506.9 mg, 1.360 mmol, 2.2 equivalents) and DIEA (0.24 mL, 1.4 mmol, 2.2 equivalents). Adjust the volume to 15 mL with anhydrous DMF. Mix in a rotary mixer for 30 minutes. Then, add the activated ester of preparation 6 to the resin and mix with a nitrogen stream for 12 hours. After 12 hours, drain the solution and wash the resin with 5 × 10 mL of DMF and 7 × 10 mL of DCM for 1 minute with N2 mixture. Dry the resin on a synthesizer for 8 hours.

[0140] Deprotection and cleavage: A 20 mL cleavage cocktail prepared with 5% w / v dithiothreitol (DTT), 2.5% v / v water, 2.5% v / v triisopropylsilane (TIPS), and 90% trifluoroacetic acid (TFA) is added to the dried resin (2.42 g) and mixed in a rotary mixer for 3 hours. The resin is filtered and washed with 2 x 2.0 mL of TFA. The filtrate is poured into 200 mL of cold MTBE and the peptide is immediately precipitated. The filtration flask is washed with 2 x 2 mL of TFA and poured into cold MTBE. After cooling to -20°C for 30 minutes, it is centrifuged. The peptide precipitate is washed twice with 240 mL of MTBE and centrifuged. The peptide precipitate is dried in a vacuum oven at 27°C for 14 hours. After drying, 1.853 g of crude preparation 34 is obtained. Purification is performed by RP-HPLC at ambient temperature on a Kromasil 100-10-C8 10 μm column (30 mm x 250 mm). A linear gradient is used, consisting of 15% acetonitrile in water for the first 5 minutes, followed by 30-55% acetonitrile in water for 25 minutes, and a constant 0.1% TFA for 30 minutes. 1.28 g of purified preparation 34 (SEQ ID NO: 33) is obtained [predicted value (mass + 2H)]. + ) / 2 = 1470.7929, observed value (mass + 2H + ) / 2 = 1470.7885].

[0141] Thioester synthesis (conversion from preparation 33 to preparation 35) Crude peptide hydrazide (Preparation 33, 2.422 g, 1.251 mmol) is dissolved in 50 mL of ligation buffer (6 M guanidine hydrochloride and 0.2 M disodium hydrogen phosphate monobase, pH 3.35) and cooled to -15°C in an acetone-ice bath. 9.4 mL of 1 M sodium nitrite solution (9.4 mmol, 7.5 equivalents) is added to the peptide hydrazide solution and stirred at -15°C for 20 minutes. Meanwhile, 1 mL of 2,2,2-trifluoroethanethiol (TFET) is added to a maximum of 10 mL in ligation buffer (6 M guanidine hydrochloride and 0.2 M disodium hydrogen phosphate monobase, pH 7.0). After 20 minutes, 10 mL of the TFET mixture is added to the peptide hydrazide solution to induce in-situ thiol lysis of peptidyl azide produced from Preparation 33. [ka]

[0142] The pH of the reaction mixture is adjusted to approximately 6.95 with a 5N sodium hydroxide solution. Peptidyl azide is subjected to thiol decomposition for 45 minutes, and the volume is reduced to 100 mL with ligation buffer (pH 7.0). The crude thioester mixture is purified by RP-HPLC at ambient temperature on a Waters X-Bridge C18 10 μm column (10 mm x 250 mm). A linear gradient is used, consisting of 10% acetonitrile in water for the first 2.8 minutes, followed by 25-42% acetonitrile in water for 25 minutes, and a constant 0.1% TFA during the 28 minutes of purification. This yields 1.03 g of TFET thioester (Preparation 35 (SEQ ID NO: 34)) [Predicted value (mass + 2H + ) / 2 = 1010.4650, observed value (mass + 2H + ) / 2 = 1010.4620].

[0143] Native Chemical Ligation: An aqueous solution of 6M guanidine hydrochloride and 0.3M disodium hydrogen phosphate mononucleotide (pH 7.0) is the ligation buffer used in native chemical ligation. All solutions are prepared with this ligation buffer. Dissolve 350.4 mg (0.174 mmol) of peptide thioester preparation 35 (SEQ ID NO: 34) in 50 mL of ligation buffer. Add 8.0 mL of 0.5 M 4-mercaptophenylacetic acid (MPAA) solution to the peptide thioester solution. Dissolve N-terminal cysteine-containing peptide (Preparation 34 (SEQ ID NO: 33), 524.6 mg, 0.178 mmol, 1.03 equivalents) in 48 mL of ligation buffer in a 50 mL centrifuge tube. Add the solution of Preparation 34 to the thioester solution. Rinse the centrifuge tube with 2 x 8 mL of ligation buffer (approximately pH 7.0) and add it to the reaction mixture. Adjust the pH of the reaction mixture to approximately 7 with 5N NaOH solution. Add 8.0 mL of tris(2-carboxyethyl)phosphine (TCEP, 0.5 M, pH 7.0) to the reaction mixture and adjust the pH again to 7.0 with 0.2 mL of 5N sodium hydroxide solution. Stir the reaction mixture at room temperature for 24 hours, then store in the freezer. Before purification, add an additional 3 mL of 0.5 M TCEP solution. Purify preparation 36 (SEQ ID NO: 35) at ambient temperature using RP-HPLC on a Kromasil C18 10 μm column (10 mm × 250 mm) for 28 minutes, with a linear gradient of 10% acetonitrile in water for the first 4 minutes and 20-50% acetonitrile in water (titrated to 0.1% acetic acid and pH 9.0) for 23 minutes. Approximately 372 mg (44.3%) of tilzepatid cysteine ​​analog preparation 36 is obtained after purification [predicted value (mass + 3H + ) / 3 = 1615.17263, observed value (mass + 3H + ) / 3 = 1615.1686]. [ka]

[0144] Desulfurization: An aqueous solution of 6-guanidine hydrochloride and 0.3 M disodium hydrogen phosphate monobase (pH 7.0) is used as the buffer for desulfurization. All solutions are prepared with this buffer. Dissolve 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (Preparation 37, 808.2 mg, 2.5 mmol) in 10 mL of buffer and adjust the pH to approximately 7.0 with 5 N NaOH. Increase the volume to 15 mL with buffer. Dissolve cysteine ​​analog Preparation 36 (105.2 mg, 0.022 mmol) in 30 mL of buffer and add 6 mL of Preparation 37 solution to it. Add 5 mL of 0.3 mL glutathione reduction solution (L-GSH, pH 7.0) and 7.5 mL of 0.5 M TCEP solution (pH 7.0). When the solution is heated at 44°C for 4.5 hours, UPLC analysis indicates that the reaction is complete [predicted value (mass + 3H)]. + ) / 3 = 1604.5153, observed value (mass + 3H + ) / 3 = 1604.5122]. The desulfurization yield is calculated by UPLC using the tilzepatide (SEQ ID NO: 1) reference standard. The yield is estimated to be 47%. [ka]

[0145] Native chemical ligation (approach 2): Synthesis of peptide hydrazide preparation 39 Sequence ID: 36 Hydrazine-CTC resin (2.03 g, 1.32 mmol, load value: 0.65 mmol / g) is placed in a 40 mL reaction vessel and swelled on a Symphony synthesizer with 3 x 10 mL DCM (30 seconds each) followed by 2 x 10 mL DMF (20 minutes each). Dissolve HBTU (1.48 g, 3.90 mmol, 3.0 equivalents) in 13.1 mL of (25S, 52S)-52-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-25-(tert-butoxycarbonyl)-2,2-dimethyl-4,23,28,37,46-pentaoxo-3,32,35,41,44-pentaoxa-24,29,38,47-tetraazatripentacontan-53-acid (Preparation 17, 365 mg / mL in DMF) solution (3.91 mmol, 3.0 equivalents). Add DIPEA (1.4 mL, 8.04 mmol, 6.1 equivalents) to the above solution and bring the volume to 19 mL with DMF. Mix the solution on a rotary mixer at room temperature for 30 minutes. The activated ester solution of preparation 17 is added to the resin. The slurry is mixed with nitrogen for 8 hours. After 8 hours, the resin is washed with 5 x 10 mL of DMF and 5 x 10 mL of DCM, and dried for 12 hours. The load of the obtained resin is measured to be 0.26 mmol / g by quantitative NMR. 1.82 g of this resin is used for the synthesis of peptide hydrazide preparation 39 (SEQ ID NO: 36).

[0146] Deprotection: 4 x 9 mL of 20% v / v piperidine in DMF, 30 minutes each.

[0147] Coupling: 3 equivalents of amino acids, 3 equivalents of OXYMA, and 3.3 equivalents of DIC are used for amino acid coupling.

[0148] After each coupling and deprotection iteration, the resin is washed with 5 × 9 mL of DMF for 1 minute with N2 mixture. After peptide hydrazide synthesis, the resin is washed with 7 × 10 mL of DCM for 1 minute with N2 mixture. The resin is then dried on a synthesizer for approximately 12 hours.

[0149] Deprotection and cleavage: A 25 mL cleavage cocktail prepared with 5% w / v dithiothreitol (DTT), 2.5% v / v water, 2.5% v / v triisopropylsilane (TIPS), and 90% trifluoroacetic acid (TFA) is added to the dried resin and mixed in a rotary mixer. The resin is filtered, washed with TFA (2 x 2.5 mL), and the filtrate is poured into 175 mL of cold MTBE. The filtration flask is washed with TFA (2 x 2.5 mL), and the washings are poured into cold MTBE. After cooling to -20°C for 30 minutes, the mixture is centrifuged. The peptide precipitate is then washed twice with 150 mL of MTBE and centrifuged. The peptide precipitate is 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) is obtained. Crude peptide hydrazide, preparation 39, is purified by RP-HPLC on a Waters XSelectCSHC18 10 μm column (10 mm x 250 mm) at ambient temperature. A linear gradient is used, consisting of 10% acetonitrile in water for the first 3 minutes, followed by 20–55% acetonitrile in water for 23 minutes, and a constant 0.1% TFA for 28 minutes of purification. Approximately 110 mg of partially purified hydrazide preparation 39 is obtained.

[0150] Deprotection and cleavage: A 25 mL cleavage cocktail prepared with 5% w / v dithiothreitol (DTT), 2.5% v / v water, 2.5% v / v triisopropylsilane (TIPS), and 90% trifluoroacetic acid (TFA) is mixed with the dried resin (2.92 g) in a rotary mixer. The resin is filtered and washed with 2 x 2.5 mL of TFA. The filtrate is poured into 200 mL of cold MTBE to immediately precipitate the peptide. The filter flask is then washed with 2 x 2 mL of TFA, and the washings are poured into cold MTBE. After cooling to -20°C for 30 minutes, the mixture is centrifuged. The peptide precipitate is then washed twice with 240 mL of MTBE and centrifuged. The peptide precipitate is 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) is obtained.

[0151] Native chemical ligation: An aqueous solution of 6 M guanidine hydrochloride and 0.3 M disodium hydrogen phosphate monobase (pH 7.0) is the ligation buffer used in native chemical ligation. All solutions are prepared with this ligation buffer. Partially purified peptide hydrazide (preparation 39, 56 mg, 0.019 mmol) is dissolved in 5 mL of ligation buffer (6 M guanidine hydrochloride and 0.3 M disodium hydrogen phosphate monobase, pH 3.35) and cooled to -15°C in an acetone-ice bath. 0.25 mL of 1 M sodium nitrite solution (0.25 mmol, 13.2 equivalents) is added to the peptide hydrazide solution and stirred at -15°C for 10 minutes. After 10 minutes, 0.8 mL of 0.5 M 4-mercaptophenylacetic acid (MPAA) solution is added to the peptide hydrazide solution to induce in-situ thiol lysis of the peptidyl azide produced from preparation 39. The pH of the reaction mixture is adjusted to approximately 7.0 with 5 N sodium hydroxide solution. The thiol lysis of peptidyl azide is carried out for 30 minutes. [ka]

[0152] Approximately 0.62 mmol of preparation 40 (SEQ ID NO: 37) is synthesized on Sieber mid resin using a standard SPPS protocol. N-terminal cysteine-containing preparation 40 (26.1 mg, 0.014 mmol, 0.74 quiv) is dissolved in 1 mL of ligation buffer. The solution of preparation 40 is added to the thioester solution. The vial containing preparation 40 is rinsed with 1 mL of ligation buffer (pH 7.0) and added to the reaction mixture. After 15 minutes, 1.0 mL of tris(2-carboxyethyl)phosphine (TCEP, 0.5 M, pH 7.0) is added to the reaction mixture, and the pH is adjusted to 7.0 with 5 N sodium hydroxide solution. The reaction mixture is stirred at room temperature for 1 hour. Tylzepatidocysteine ​​analog preparation 42 is observed in the reaction mixture. [ka]

[0153] array Sequence ID 1 Chilzepatid YX1EGTFTSDYSIX2LDKIAQKAFVQWLIAGGPSSGAPPPS In the formula, X1 is Aib, X2 is Aib, and the K at position 20 is bonded to the epsilon-amino group of the K side chain (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γGlu)1-CO-(CH2) 18 Chemically modified with -CO2H, the C-terminal amino acid is amidated as a C-terminal primary amide. Sequence ID 2 [ka] Sequence ID 3 [ka] Sequence ID 4 [ka] Sequence ID 5 [ka] Sequence ID 6 [ka] Sequence ID 7 [ka] Sequence ID 8 [ka] Sequence ID 9 [ka] Sequence ID 10 [ka] Sequence ID 11 [Chemical formula] Sequence number 12 [Chemical formula] Sequence number 13 [Chemical formula] Sequence number 14 [Chemical formula] Sequence number 15 [Chemical formula] Sequence number 16 [Chemical formula] Sequence number 17 [Chemical formula] Sequence number 18 [Chemical formula] Sequence number 19 [Chemical formula] Sequence number 20 [Chemical formula] Sequence number 21 [Chemical formula] Sequence number 22 [Chemical formula] Sequence number 23 [Chemical formula] Sequence number 24 [Chemical formula] Sequence number 25 [Chemical formula] Sequence number 26 [Chemical formula] Sequence number 27 [Chemical formula] Sequence number 28 [Chemical formula] Sequence number 29 [Chemical formula] Sequence number 30 [Chemical formula] Sequence number 31 [Chemical formula] Sequence number 32 [Chemical formula] Sequence number 33 [Chemical formula] Sequence number 34 [Chemical formula] Sequence number 35 [Chemical formula] Sequence number 36 [ka] Sequence ID 37 [ka] Sequence ID 38 [ka] Sequence ID 39 [ka] Sequence ID 40 [ka]

Claims

1. The compound of Sequence ID No. 17, or a pharmaceutically acceptable salt thereof.

2. The compound of Sequence ID No. 11, or a pharmaceutically acceptable salt thereof.

3. The compound of SEQ ID NO: 22, or a pharmaceutically acceptable salt thereof.

4. The compound of SEQ ID NO: 21, or a pharmaceutically acceptable salt thereof.

5. The compound of Sequence ID No. 20, or a pharmaceutically acceptable salt thereof.

6. The compound of the following formula, 【Chemistry 1】 or a pharmaceutically acceptable salt thereof.

7. The compound of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.

8. The compound of the following formula, 【Chemistry 2】 or a pharmaceutically acceptable salt thereof.

9. The compound of SEQ ID NO: 4, or a pharmaceutically acceptable salt thereof.

10. The compound of Sequence ID No. 7 or a pharmaceutically acceptable salt thereof.

11. The compound of Sequence ID No. 14, or a pharmaceutically acceptable salt thereof.

12. The compound of Sequence ID No. 17, or a pharmaceutically acceptable salt thereof.

13. The compound of Sequence ID No. 33, or a pharmaceutically acceptable salt thereof.

14. The compound of SEQ ID NO: 32, or a pharmaceutically acceptable salt thereof.

15. The compound of Sequence ID No. 34, or a pharmaceutically acceptable salt thereof.

16. The compound of Sequence ID No. 35, or a pharmaceutically acceptable salt thereof.

17. The compound of sequence number 36, or a pharmaceutically acceptable salt thereof.

18. The compound of Sequence ID No. 38, or a pharmaceutically acceptable salt thereof.

19. The compound of sequence number 39, or a pharmaceutically acceptable salt thereof.

20. A process for preparing tilzepatide or a pharmaceutically acceptable salt thereof, including nanofiltration of the intermediate tilzepatide.

21. The process according to claim 20, wherein the intermediate tilzepatide is Sequence ID No.

27.

22. The process according to claim 20, wherein the intermediate tilzepatide is Sequence ID No.

29.

23. The process according to any one of claims 20 to 22, wherein the nanofiltration process includes diafiltration.

24. The process according to any one of claims 20 to 23, wherein the process includes DMF diafiltration.

25. A process for preparing tilzepatide or a pharmaceutically acceptable salt thereof, comprising deprotecting the compound of SEQ ID NO: 22 or a pharmaceutically acceptable salt thereof.

26. The process according to claim 25, wherein the deprotection solution comprises dithiothreitol, triisopropylsilane, and trifluoroacetic acid.

27. A process for selectively acylating lysine amino acids in a peptide, wherein the peptide-lysine-NH bonded to the resin ２ A process comprising coupling with t-butyl-eicosanedioyl-Glu-(O-tert-butyl)-(8-amino-3,6-dioxaoctanoic acid)-(8-amino-3,6-dioxaoctanoic acid)-OH.

28. The process according to claim 27, wherein the peptide is an incretin.

29. Peptide-lysine-NH bonded to the aforementioned resin ２ The process according to any one of claims 27 or 28, wherein the solution is sequence number 24, or a pharmaceutically acceptable salt thereof.

30. A process for converting a depsipeptide isomer into a desired peptide, a. Adjusting the pH of the depsipeptide isomer to approximately pH 7 to approximately pH 10, b. A process comprising incubating the depsipeptide isomer at pH 7 to pH 10 for at least 1 hour.

31. The process according to claim 30, wherein the depsipeptide isomer is adjusted to a pH of approximately 8.5 to approximately 9.

5.

32. The process according to any one of claims 30 and 31, wherein the peptide is an incretin.

33. The process according to any one of claims 30 to 32, wherein the depsipeptide isomer is recovered from the process waste stream.

34. The process according to any one of claims 30 to 33, wherein the depsipeptide isomer is SEQ ID NO: 40 or a pharmaceutically acceptable salt thereof.

35. A process for desulfurizing an incretin or a pharmaceutically acceptable salt thereof, comprising contacting the incretin with a radical initiator.

36. The process according to claim 35, wherein the radical initiator is a water-soluble radical initiator.

37. The process according to any one of claims 35 or 36, wherein the radical initiator is an azo initiator.

38. The process according to any one of claims 35 to 37, wherein the radical initiator is selected from the group consisting of 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride and 2,2'-azobis(2-methylpropionamidine) dihydrochloride.

39. The process according to any one of claims 35 to 38, wherein the incretin is SEQ ID NO: 35 or a pharmaceutically acceptable salt thereof.

40. The process according to any one of claims 35 to 39, wherein the desulfurization process provides the peptide of Sequence ID No. 1.

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