Process for preparing GIP / GLP1 dual agonists

Abstract

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

Description

[Technical Field]

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

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

[0003] U.S. Patent No. 9,474,780 generally describes methods for producing peptides and GIP / GLP1 dual agonists. International Publication No. 2020 / 159949 describes a method for producing tirzepatide. However, improvements to the linear process are needed to facilitate large-scale commercial production.

[0004] Processes and intermediates are needed to enable improved technologies for the production of tirzepatide with a combination of advantages, including commercially desirable purity and efficiency. Similarly, efficient, environmentally "green" processes are needed, including stable intermediates to provide tirzepatide with fewer purification steps. Improved technologies are also needed to provide tirzepatide manufacturing processes that minimize waste streams to enhance both environmental and operator safety. Large-scale preparation of pharmaceutically acceptable tirzepatide presents several technical challenges that can affect overall yield and purity. Processes are needed to avoid the use of transition metals and / or harsh reaction conditions that are incompatible with peptide synthesis.

[0005] The present invention seeks to fulfill these needs by providing novel intermediates and processes useful in the production of tirzepatide (SEQ ID NO: 5), or a pharmaceutically acceptable salt thereof. The improved tirzepatide production process of the present invention embodies a combination of advances, including an efficient route with fewer steps, while providing intermediates and process reactions that maintain high quality and purity. Importantly, the improved process and intermediates reduce resource intensity and minimize waste streams. Summary of the Invention

[0006] The improved process described herein provides various embodiments of intermediates useful in the production of tirzepatide.

[0007] The present invention provides a compound of SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof. The present invention provides a compound of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof. The present invention provides a compound of SEQ ID NO: 3, or a pharmaceutically acceptable salt thereof.

[0008] The present invention provides a process for preparing tirzepatide, comprising preparing a deprotected compound of SEQ ID NO: 1 or a pharmaceutically acceptable salt of the compound. The present invention provides a process for preparing tirzepatide, comprising deprotecting the compound of SEQ ID NO: 1 or a pharmaceutically acceptable salt of the compound. The present invention provides a process for preparing tirzepatide, comprising cleaving the compound of SEQ ID NO: 2 or a pharmaceutically acceptable salt of the compound from the resin. The present invention provides a process for preparing tirzepatide, comprising deprotecting the compound of SEQ ID NO: 3 or a pharmaceutically acceptable salt of the compound. The present invention provides a process for preparing tirzepatide (SEQ ID NO: 5), comprising acylating lysine to form the compound of SEQ ID NO: 4 or a pharmaceutically acceptable salt of the compound, followed by deprotection to form tirzepatide.

[0009] A process is provided for preparing the mtt-protected epsilon amino group of the lysine amino acid of the resin-bound, protected peptide of SEQ ID NO: 1. A process is provided for selectively deprotecting the mtt-protected epsilon amino group of the lysine amino acid of the resin-bound, protected peptide of SEQ ID NO: 1 to form the protected peptide of SEQ ID NO: 2. A process is provided for selectively cleaving the resin-bound, protected peptide of SEQ ID NO: 2 to form the compound of SEQ ID NO: 3. A process is provided for acylating the unprotected epsilon amino group of the lysine of SEQ ID NO: 3 with compound 1 to form the compound of SEQ ID NO: 4.

[0010] A process is provided for determining chiral purity for the preparation of peptides by SPPS by using a chiral high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS / MS) method. DETAILED DESCRIPTION OF THE INVENTION

[0011] In one embodiment, there is provided a process for preparing tirzepatide (SEQ ID NO: 5), comprising: a) deprotecting the compound of SEQ ID NO: 1 to provide the compound of SEQ ID NO: 2; b) removing the resin to provide the compound of SEQ ID NO:3; c) coupling SEQ ID NO:3 with Compound 1 to provide SEQ ID NO:4; d) deprotecting SEQ ID NO:4 to provide tirzepatide (SEQ ID NO:5).

[0012] In one embodiment, there is provided a process for preparing tirzepatide (SEQ ID NO: 5), comprising: a) deprotecting the compound of SEQ ID NO: 1 to provide the compound of SEQ ID NO: 2; b) coupling SEQ ID NO:2 with Compound 1 to provide SEQ ID NO:6; c) deprotection and removal of the resin to give tirzepatide (SEQ ID NO: 5). d) removing SEQ ID NO:6 from the resin to provide SEQ ID NO:4; e) deprotecting SEQ ID NO: 4 to obtain tirzepatide (SEQ ID NO: 5).

[0013] In one embodiment, there is provided a process for preparing tirzepatide (SEQ ID NO: 5), comprising: a) deprotecting the compound of SEQ ID NO: 1 to provide the compound of SEQ ID NO: 2; b) coupling SEQ ID NO:2 with Compound 1 to provide SEQ ID NO:6; c) removing SEQ ID NO:6 from the resin to provide SEQ ID NO:4; d) deprotecting SEQ ID NO:4 to obtain tirzepatide (SEQ ID NO:5).

[0014] In one embodiment, there is provided a continuous flow process for preparing tirzepatide (SEQ ID NO: 5), comprising: a) deprotecting the compound of SEQ ID NO: 1 to provide the compound of SEQ ID NO: 2; b) removing the resin to provide the compound of SEQ ID NO: 3, optionally with in-line nanofiltration to provide purification and solvent exchange; c) coupling SEQ ID NO:3 with Compound 1 to provide SEQ ID NO:4, wherein nanofiltration is arranged in-line to provide purification and solvent exchange; d) deprotecting SEQ ID NO: 4 to provide tirzepatide (SEQ ID NO: 5), wherein nanofiltration is arranged in-line to provide purification and solvent exchange.

[0015] In one embodiment, there is provided a continuous flow process for preparing tirzepatide (SEQ ID NO: 5), comprising: a) deprotecting the compound of SEQ ID NO: 1 to provide the compound of SEQ ID NO: 2; b) coupling SEQ ID NO:2 with Compound 1 to provide SEQ ID NO:6; c) removing SEQ ID NO:6 from the resin to provide SEQ ID NO:4; d) deprotecting SEQ ID NO: 4 to obtain tirzepatide (SEQ ID NO: 5), wherein nanofiltration is arranged in-line to provide purification and solvent exchange.

[0016] In one embodiment for improving efficiency, a continuous flow process and chromatographic purification for preparing tirzepatide (SEQ ID NO: 5) comprising: a) deprotecting the compound of SEQ ID NO: 1 to provide the compound of SEQ ID NO: 2; b) removing the resin to provide the compound of SEQ ID NO: 3, optionally using in-line chromatography and nanofiltration to purify and provide solvent exchange. c) coupling SEQ ID NO:3 with Compound 1 to provide SEQ ID NO:4, wherein nanofiltration is arranged in-line to provide purification and solvent exchange. d) deprotecting SEQ ID NO: 4 to provide tirzepatide (SEQ ID NO: 5), wherein chromatographic nanofiltration is arranged in-line to purify and provide solvent exchange, and chromatographic purification comprising:

[0017] In one embodiment, a continuous flow process and chromatographic purification for preparing tirzepatide (SEQ ID NO: 5) comprising: a) deprotecting the compound of SEQ ID NO: 1 to provide the compound of SEQ ID NO: 2; b) removing the resin to provide the compound of SEQ ID NO:3, and purifying and solvent exchanging the compound using in-line chromatography and nanofiltration. c) coupling SEQ ID NO:3 with Compound 1 to provide SEQ ID NO:4, wherein nanofiltration is arranged in-line to provide purification and solvent exchange. d) deprotecting SEQ ID NO: 4 to provide tirzepatide (SEQ ID NO: 5), wherein chromatographic nanofiltration is arranged in-line to provide purification and solvent exchange, and chromatographic purification.

[0018] A process for selectively acylating unprotected lysine amino acids is provided. A process for selectively acylating lysine amino acids in a peptide is provided, comprising coupling resin-bound peptide-lysine-NH2 with t-butyl-eicosanediol-Glu-(O-tert-butyl)-(8-amino-3,6-dioxaoctanoic acid)-(8-amino-3,6-dioxaoctanoic acid)-OH. A process for preparing tirzepatide is provided, comprising deprotecting the compound of SEQ ID NO: 4, or a pharmaceutically acceptable salt thereof.

[0019] The present invention provides a process for preparing tirzepatide, comprising cleaving the compound of SEQ ID NO: 2, or a pharmaceutically acceptable salt of the compound, from a resin. The present invention provides a process for preparing tirzepatide, comprising deprotecting the compound of SEQ ID NO: 3, or a pharmaceutically acceptable salt of the compound. The present invention provides a process for preparing tirzepatide, comprising acylation of lysine to form the compound of SEQ ID NO: 4, or a pharmaceutically acceptable salt of the compound.

[0020] 1. A process for selectively acylating lysine amino acids, comprising:

[0021] [ka] or a pharmaceutically acceptable salt thereof.

[0022] The improved process using a lysine MTT-protected intermediate provides orthogonal protection with higher purity than similar intermediates used in known processes. Furthermore, the synthesis of tirzepatide using an on-resin MTT-protected intermediate involves orthogonal deprotection at lysine 20, removal of MTT, and coupling with (S)-22-(tert-butoxycarbonyl)-45,45-dimethyl-10,19,24,43-tetraoxo-3,6,12,15,44-pentaoxa-9,18,23-triazahexatetracontanoic acid to provide the desired purity. Furthermore, SEQ ID NO:6 can be cleaved from the resin to form SEQ ID NO:4, which can then be deprotected to provide tirzepatide, enabling continuous flow synthesis.

[0023] Both tirzepatide synthesis processes using MTT result in improved purity and yield compared to known linear processes. Furthermore, the processes herein potentially have the added advantage that the amount of (S)-22-(tert-butoxycarbonyl)-45,45-dimethyl-10,19,24,43-tetraoxo-3,6,12,15,44-pentaoxa-9,18,23-triazahexatetracontanoic acid can be reduced stoichiometrically to approximately 1 equivalent due to LPPS conditions, potentially making them adaptable to flow synthesis methods. Reducing the need for excess (S)-22-(tert-butoxycarbonyl)-45,45-dimethyl-10,19,24,43-tetraoxo-3,6,12,15,44-pentaoxa-9,18,23-triazahexatetracontanoic acid may be environmentally and commercially important for large-scale peptide synthesis.

[0024] Determining chiral purity is important for assessing the quality of peptide pharmaceuticals. For synthetic peptides, undesired D-isomers can be introduced as impurities in the amino acid starting material and can also form during peptide synthesis and, in some cases, during product shelf life. A chiral high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS / MS) method is described that facilitates rapid and accurate determination of amino acid chiral purity in peptides. Peptides are hydrolyzed in deuterated acid to facilitate correction for any racemization that occurs during this sample preparation step, and the amino acids are subsequently separated by chiral chromatography coupled with ESI-MS / MS for quantification. Amino acid samples are analyzed directly after hydrolysis by chromatographic separation and extraction of selected ion responses, offering efficiency and simplicity by avoiding the derivatization step required by conventional methodologies. The feasibility of method validation is described for all 19 chiral proteinogenic amino acids, and practical application is demonstrated for the analysis of model peptides. The method was proven capable of quantitative determination of trace levels of D-isomer impurities over the desired range of 0.1% to 1.0%.

[0025] As used herein, the following abbreviations have the meanings set forth herein: "SPPS (Solid Phase Peptide Synthesis)" means solid phase peptide synthesis, "MTT (4-methyltrityl)" and / or "mtt" means 4-methyltrityl, "Fmoc (fluorenylmethyloxycarbonyl chloride)" means fluorenylmethyloxycarbonyl chloride, "Pip (piperidine)" means piperidine, "DIC (diisopropylcarbodiimide)" means diisopropylcarbodiimide, "Oxyma" means ethyl cyanohydroxyiminoacetate, "DCM (dichloromethane)" means dichloromethane, "IPA" means isopropanol, "MTBE (methyl-tert-butyl ether)" means methyl-tert-butyl ether, "TFA (trifluoroacetic acid)" means methyl tert-butyl ether, "TIPS (triisopropylsilane)" means trifluoroacetic acid, "DTT (dithiothreitol)" means dithiothreitol, "UPLC (Ultra High Performance Liquid Chromatography)" means ultra-high performance liquid chromatography, "HFIP (hexafluoroisopropanol)" means hexafluoroisopropanol, "CTC" means chlorotrityl, "HATU" means (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, "TFET (2,2,2-trifluoroethanethiol)" means 2,2,2-trifluoroethanethiol, "DIEA (N,N-diisopropylethylamine)" means N,N-diisopropylethylamine, and "AEEA" means 17-amino-10-oxo-3,6,12,15-tetraoxa-9-azaheptadecanoic acid, "TCEP (tris(2-carboxyethyl)phosphine)" means tris(2-carboxyethyl)phosphine, "DCU (dicyclyhexylurea)" means dicyclhexylurea, "DCC (dicyclhexylcarbodiimide)" means dicyclohexylcarbodiimide, "TMSA (trimethylsilyalmide)" means trimethylsilylamide, "HOBt (hydroxybenzotriazole)" means hydroxybenzotriazole, "HRMS (high resolution mass spectrometry)" means high resolution mass spectrometry, "LPPS (liquid phase peptide synthesis)" means liquid phase peptide synthesis, "MSMPR (mixed product mixed suspension reactor)" means mixed product mixed suspension reactor, "MPA (mobile phase A)" means mobile phase A, "MPB (mobile phase "B)" means mobile phase B, "L-GSH" means L-glutathione reducing solution, "TZP (tirzepatide)" means tirzepatide, "AP (active pharmaceutical)" means active pharmaceutical ingredient, "API (active pharmaceutical ingredient)" means active pharmaceutical ingredient, "PyBOP" means (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, "DEA (diethylamine)" means diethylamine, "TBTU" means 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate, "TNTU" means 2-(5-norbornene-2,3-dicarboximide)-1,1,3,"PyClock" refers to 3-tetramethyluronium tetrafluoroborate, "PyOxim" refers to 1-cyano-2-ethoxy-2-oxoethylideneaminooxy-tris-pyrrolidino-phosphonium hexafluorophosphate, and "PyClock" refers to 6-chloro-benzotriazol-1-yloxy-tris-pyrrolidinophosphonium hexafluorophosphate. As presented herein, single-letter amino acid abbreviations are presented in bold, and atoms are presented as non-bold text, generally in a smaller font, to distinguish them from single-letter amino acid abbreviations. As used herein, when an amino acid abbreviation appears with a number above the amino acid, that number refers to the position of the corresponding amino acid in the final tirzepatide product. Numbers are provided for convenience, and the presence or absence of such numbers in a sequence does not affect the amino acid sequence or peptide depicted in such sequence. As used herein, the term "protected" means that a protecting group is attached at the indicated position. Those skilled in the art will recognize that a variety of protecting groups are well known, and alternative protecting groups may be suitable for a particular process. As used herein, "in-line" refers to continuous flow synthesis, where the synthesis is provided with the mentioned characteristics so that the synthetic process can continue without interruption. In some embodiments, in-line nanofiltration may be used for purification and / or solvent exchange, as desired in the synthesis. In some embodiments, in-line chromatography may be used in-line during the synthetic process.

[0026] Those skilled in the art will appreciate that alternative resins exist for constructing the peptides presented herein. For example, Sieber and Rink amide resins are well known to those skilled in the art for preparing the peptides disclosed herein. However, alternative resins may be selected for preparing the peptides described herein. For example, but not limited to, 2-CTC and related resins can be used to prepare the target peptides, followed by a C-terminal amidation step.

[0027] The processes described herein are amenable to nested steps, e.g., multiple process steps running simultaneously and involving multiple unit operations, allowing for the assembly of multiple pre-assembled peptide units via continuous unit operations or "continuous flow." Continuous flow processes are enabled by fume hood designs that support continuous flow. Continuous flow synthesis can enable safe, cost-effective, and efficient continuous production to meet commercial demand for peptides.

[0028] The processes provided herein can be performed using nanofiltration for purification and solvent exchange, eliminating the need for additional isolation steps and enabling a continuous processing platform.

[0029] A unit operation-based continuous manufacturing skid located in a fume hood can be used to carry out peptide unit coupling reactions in flow. Feeds for peptide unit coupling reactions are precisely controlled to achieve high reaction conversions while maintaining strict stoichiometric control, regardless of expected variability from solid-phase synthesized fragments. Feed flow rates for continuous flow processes can be automatically updated as needed for the process.

[0030] Nanofiltration can be implemented for purification and solvent exchange of coupled peptide solutions. Nanofiltration can eliminate the isolation step of amorphous solids and allow for a fully nested process throughout the peptide unit reaction. A standby nanofiltration skid can allow for continued production during scheduled maintenance of the nanofiltration skid.

[0031] The peptide solution can be precipitated and isolated in a dual-train batch precipitation and isolation system. Once the crude peptide is assembled, the resulting peptide solution can be precipitated and isolated using a batch precipitation and isolation system. The dual-train system can allow for a continuous flow reaction to continue as the batch precipitation and isolation unit operation is performed. A pack-off barrier can be used to enclose the O-ring canister packaging technology to protect both the operator and the facility.

[0032] Equipment used in synthetic peptide manufacturing processes can be equipped with cleaning-in-place circuits and cleaning batch recipes to ensure that cleaning is convenient, efficient, and effective.

[0033] Solid-phase synthesis allows for the isolation of growing peptide fragments, alleviating the need for isolation of amorphous intermediates and the necessary by-product purging and washing steps that may be required to enable successive assembly. Conventional solution-phase couplings generally require complex isolation steps to purge such by-products. Washing of the coupled peptide while in solution phase may be used to eliminate the complexity of intermediate isolation between couplings of individual units.

[0034] Nanofiltration can provide a means for concentrating, washing, and exchanging solvents after the coupling process. Because the polar aprotic solvents required for peptide solubility are challenging, unconventional ceramic membranes may offer a useful alternative to more typical polymeric membranes.

[0035] Nanofiltration can provide an efficient, scalable, and cost-effective method for integrating multiple sequential coupling steps into a complete peptide assembly system. Nanofiltration technology enables the use of continuous synthesis platforms because it eliminates the need for intermediate isolation during time-consuming process steps. Nanofiltration can be effective for peptides of a wide range of molecular weights, allowing the platform to be applicable to multiple peptide projects.

[0036] Ceramic membranes are made from inorganic materials such as, but not limited to, alumina, zirconia oxide, and silicon carbide. The inorganic materials used to make ceramic membranes allow for use with aggressive media, such as acids and strong solvents, which polymeric membranes cannot, making them suitable for use in the liquid-phase coupling reactions described herein. Ceramic membranes can be made in a tubular cross-flow configuration suitable for use in the continuous coupling steps described herein. Ceramic membranes are available from a variety of suppliers, including, but not limited to, Altech (Gladbeck, Germany), CoorsTek (Golden, Colorado), Inopor (ScheBlitz, Germany), Mantec Technical Ceramics Ltd (Staffordshire, England), and Orelis Environnement / Alsys group (Salindres, France).

[0037] The underivatized amino acid methodology, used with a stationary phase, can be optimized for determining chiral purity through the quantification of trace levels of D-isomers in synthetic peptides. Samples were hydrolyzed in deuterium chloride (DCl) so that racemized amino acids could be excluded by the mass spectrometric detector during the sample preparation process. Eliminating sample derivatization from the process can facilitate a simplified method with increased throughput potential. Data are presented to demonstrate chiral HPLC-ESI-MS / MS analysis of a full mixture of 19 chiral D / L natural amino acid pairs, with quantification of D-isomers achieved by monitoring extracted ion chromatograms (EICs) associated with peptide ions specific to each amino acid. The feasibility of the method for quantification of trace levels of D-isomers in L-isomer matrices is demonstrated through determination of linearity, accuracy, precision, and detection / quantitation limits, supporting synthetic peptide manufacturing process control limits of 0.5% and method reporting limits of 0.1% for each D-isomer, consistent with pharmaceutical industry regulatory guidance for synthetic peptides. The chiral HPLC-ESI-MS / MS method was validated through application to the analysis of four model peptides, each consisting of 8 to 14 amino acid residues.

[0038] The four model peptides used in this study consist of 8–14 amino acid residues each and are listed by amino acid content along with residue number in Table 1. L- and D-isomer peptide materials were synthesized as highly pure amorphous solids. The peptides were fully characterized for identity, purity, and content. Samples were synthesized as "protected" peptides in which the active side chains of the amino acids (e.g., hydroxyl, carboxyl, amino) were protected with functional groups such as Fmoc, trityl (Trt), tert-butyloxycarbonyl (Boc), and tertiary butyl (tBu), as listed in Table 1. Chiral purity analysis of the peptide samples was also performed using conventional GCMS and LCMS methodologies for comparison.

[0039] [Table 1] * Aib (2-aminoisobutyric acid) is a non-chiral, non-proteinogenic amino acid

[0040] [ka]

[0041] Sample preparation The nominal target concentration of each amino acid was 0.1 mg / mL. Amino acid standards were prepared in water, except for aspartic acid, cysteine, and tyrosine, which required approximately 4% formic acid for solubility (these three amino acids were first dissolved in formic acid and then diluted to volume with water). Amino acid standard solutions were prepared at concentrations ranging from approximately 0.0005 mg / mL to 0.002 mg / mL, representing a range of 0.05 to 2.0% nominal amino acid concentrations in the peptide hydrolysis mixture.

[0042] For analysis of Fmoc-protected peptide fragments B and C, samples were incubated in piperidine solution (3 mL piperidine + 10 mL dichloromethane) at a concentration of approximately 1 mg / mL (to provide a minimum amino acid concentration of 0.1 mg / mL for 10-amino acid peptides) for 15 min at 2–8 °C to remove the Fmoc protecting group prior to hydrolysis. After Fmoc removal, samples were dried under a stream of nitrogen for 4 h. All dried peptide samples were digested by acid hydrolysis in 1.0 mL of 5 N DCl at a concentration of approximately 1 mg / mL in 10 mL glass vials with Teflon heat-resistant caps at 110 °C for 4 h. To prevent oxidation of tryptophan to N'-formylkynurenine or related species, a sample of peptide fragment B was mixed with 5 N DCl + 1% phenol, purged with nitrogen, and then carefully sealed with Teflon tape and parafilm around the vial cap prior to hydrolysis at 110 °C. In addition to cleaving the peptide into its individual amino acid components, remaining protecting groups (tBu, Boc, and Trt) are removed during the hydrolysis step. After hydrolysis, the sample was cooled to ambient temperature and 2 mL of water was added. The sample was then dried for 12 hours using a Genevac™ EZ-2 Elite HCl-compatible centrifugal vacuum concentrator (Genevac Ltd. Ipswitch, UK), and the acid was removed by lyophilization. As a final step, the sample was reconstituted in water to the original 1 mL volume and then analyzed by chiral HPLC-ESI-MS / MS. Samples for proline analysis using the Chiralpak® ZWIX(-) column method were further diluted 1:1 with water before injection.

[0043] Chiral HPLC-ESI-MS / MS The UPLC system included a binary solvent manager and an autosampler. Separations were performed on two Crownpak® CR-I(+) 150 × 3.0 mm ID columns. Two 5 μm columns (Chiral Technologies, Inc., West Chester, PA) were connected in series using isocratic elution with a mobile phase composition of 5% A (0.5% TFA in water) and 95% B (85 ACN / 15 EtOH / 0.5 TFA; v / v / v). The flow rate was 0.4 mL / min, the injection volume was 1 μL, and the column temperature was 30°C. Separations were also performed using a single Chiralpak® ZWIX(-) 150 × 3.0 mm ID column. A 5 μm column (Chiral Technologies, Inc. West Chester, PA) and the mobile phase used with this column was isocratic and consisted of 25 mM formic acid, 25 mM ammonium formate in methanol / water (98 / 2 v / v).

[0044] The UPLC was coupled to a mass spectrometer equipped with a Turbo Spray IonDrive operated in multiple reaction monitoring (MRM) mode. Separation used the chromatographic conditions described above, with the diversion valve programmed to divert the first 2 minutes to waste. The mass spectrometer was operated in positive ion polarity, with the ion spray voltage set at 5500 V and the temperature set at 550 °C. The MRM transitions for each amino acid can be found in Table 2, along with the respective Q1 and Q3 m / z values ​​(both set to unit resolution), as well as the optimized declustering potential, entrance potential, collision energy, and collision cell exit potential values. The 132.1 to 86.0 m / z transition represents both Leu and Ile as both amino acid peptides in this format; this transition does not provide any specificity between this isobaric amino acid pair. However, chromatography can effectively separate Leu and Ile isomers (20, 25). In addition, the transition from 132.1 to 69.0 m / z is specific to isoleucine and provides a transition that can be used for isoleucine quantification. Other relevant instrument parameters included curtain gas at 20 PSI, ion source gas 1 at 40 PSI, ion source gas 2 at 60 PSI, and collision gas set to low. Data acquisition and processing were performed using Analyst software (SCIEX).

[0045] [Table 2]

[0046] Chiral HPLC separation of amino acids Both crown ether and quinine / quinidine-derived zwitterionic stationary phases used in hydrophilic interaction chromatography (HILIC) mode have proven to be highly effective chiral selectors for the separation of amino acid enantiomers. The crown ether of the Crownpak® stationary phase strongly interacts with the protonated amino groups in the strongly acidic TFA mobile phase, and the binaphthyl moiety then facilitates stereospecific retention due to the structural differences between the amino acid enantiomers. Crown ethers provide separation of all D / L amino acid pairs except D / L proline due to their low affinity for the amino group of secondary amines. Proline chiral separation is instead provided by Chiralpak® ZWIX, an alkaloid-derived zwitterionic stationary phase that is believed to induce selectivity through ion pairing in mobile phases containing both acidic and basic additives. The elution order of the separation of isomeric analytes provided by both of these packings can be tailored by selecting the appropriate chiral configuration of the stationary phase. The Crownpak® CR-I(+) and Chiralpak® ZWIX(-) packings were specifically selected for their ability to facilitate elution of the D-isomer before the L-isomer, thereby minimizing any interference due to tailing of the L-isomer peak, which is present in higher concentrations.

[0047] A chiral chromatography method using Crownpak® CR-I(+) and Chiralpak® ZWIX(-) columns was designed by referencing parameters previously described for the analysis of D-amino acids in food. However, in the case of the current study, two Crownpak® CR-I(+) columns were connected in series to provide optimal resolution. For the determination of D-isomer impurities in synthetic peptides, chromatographic separation was combined with a high-low sample concentration chromatography strategy (26). High-low chromatography derives its name from the use of paired sample solutions, with "high" corresponding to a nominal sample concentration of approximately 0.1 mg / mL for each individual L-amino acid in the hydrolysis solution mixture, and "low" representing a sample concentration of approximately 0.001 mg / mL. The calculation of the % D-isomer is based on a comparison of the chromatograms obtained from the two solutions, with the peak area of ​​the L-isomer present in the low concentration sample effectively serving as an external standard calibration for the area % quantification of the D-isomer peak area measured in the high concentration sample:

[0048]

number

[0049] Chiral HPLC-ESI-MS / MS analysis of amino acids In chiral HPLC-ESI-MS / MS analysis, the protonated molecular ion ([M+H] +) are selected as precursor ions for all target compounds based on the expected m / z values ​​corresponding to each unracemized (i.e., non-deuterated) amino acid in DCl. Peptidation is then induced, and the ion signal response of the product ions specific to each amino acid is measured. The precursor and product ions used for quantification of each amino acid are listed in Table 5. After sample preparation and analysis, the extracted ion chromatograms (EICs) associated with the product ion responses associated with each amino acid represent the D- and L-amino acids essentially present in the peptide before hydrolysis. As described in the introduction, any amino acids that racemize during hydrolysis are excluded as precursor ions due to the increase in molecular mass of one atomic mass unit resulting from the incorporation of deuterium. Using this approach, all amino acids present in a peptide can be analyzed after hydrolysis in a single chiral HPLC-ESI-MS / MS experiment by monitoring the EICs corresponding to the expected product ions eluting at specific chromatographic retention times.

[0050] To confirm the sensitivity, linearity, and accuracy supporting the method reporting limit of 0.1% and the quantification range of D-isomers from 0.1% to 1.0%, a series of standards were prepared and analyzed for each of the amino acids at six levels (0.05%, 0.1%, 0.2%, 0.5%, 1.0%, and 2.0%). For all amino acids, the detector response was such that a response at 0.1% produced an S / N > 10 and linearity was < R 2 The method demonstrated excellent sensitivity, with a ρ = 0.9900 (see Table 3). D-isomer spikes were also prepared in the corresponding L-isomer matrix. Spiked samples were introduced at a nominal concentration of 0.1 mg / mL into the corresponding L-isomer matrix at levels of 0.1%, 0.2%, 0.5%, and 1.0%. The method's ability to determine D-isomer responses with an accuracy of approximately 80-120% recovery was demonstrated; the percent recovery was based on a metered spiked sample preparation and spike responses directly compared to the corresponding standard curve. Sensitivity, linearity, and accuracy data representing all 19 chiral amino acids are listed in Table 3, and the results support the quantitative capability of this method.

[0051] [Table 3]

[0052] Batches of all four model peptides were analyzed by both chiral HPLC-ESI-MS / MS and conventional methodologies for comparison and confirmation, and are reported. GC-MS with derivatization was utilized for comparative studies of peptides A and D, while HPLC-MS with derivatization was used for comparative studies of peptides B and C. The results are shown in Table 4 and demonstrate the expected low levels of D-isomers (approximately + / - 0.1% based on 3 × SD for the validation of the current and GC-MS methods (30) in the 0.1–0.2% D-isomer range), with good agreement between the new and conventional techniques, within the inherent variability estimated based on the accuracy and precision associated with each method.

[0053] [Table 4] 1 Testing of peptides A and D was carried out essentially as described by R. Liardon, S. Ledermann, and U. Ott, "Determination of D-amino acids by deuterium labeling and selected ion monitoring," J. Chromatogr. A 203 (1981) 385-395. 2 Testing of peptides B and C was carried out essentially as described in K. Kalikova, T. Slechtova, and E. Tesarova, "Enantiomeric ratio of amino acids as a tool for determination of aging and disease diagnostics by chromatographic measurement," Separations 3 (2016) 30-48.

[0054] Solid-phase peptide synthesis (SPPS) construction is achieved using standard fluorenylmethyloxycarbonyl chloride (Fmoc) peptide chemistry with sequential couplings on an automated peptide synthesizer. The resin is swollen in DMF and then deprotected using 20% ​​piperidine (Pip) / DMF (3 x 30 min). Subsequent Fmoc deprotection uses 3 x 30 min treatments of 20% Pip / DMF, with 4 x 30 min treatments used for more difficult couplings. After deprotection, the resin is washed with 5 x 2 min washes of 10 volumes of DMF. Amino acid preactivation uses diisopropylcarbodiimide (DIC) / ethyl cyanohydroxyiminoacetate (Oxyma) DMF solution for 30 min at room temperature. Coupling of activated amino acids to the resin-bound peptide occurs for the time specified for each individual amino acid. Each coupling is followed by 5 x 2 min solvent washes with 10 volumes of DMF. To isolate the final product, the resin-bound product is washed with 10 volumes of DCM for 5 × 2 min to remove DMF. The resin is washed with 10 volumes of IPA for 2 × 2 min to remove DCM, and then with 10 volumes of methyl tert-butyl ether (MTBE) for 5 × 2 min. The product is then vacuum dried at 40 °C. The resin-bound product is stored refrigerated (-20 °C). For analysis, the peptide is cleaved from the resin with an acidic cocktail consisting of trifluoroacetic acid (TFA), HO, TIPS (triisopropylsilane), and DTT (dithiothreitol) in the following ratio (0.93 v / 0.04 v / 0.03 v / 0.03 w). The resin is swelled with DCM (4–5 mL, 3 × 30 min) 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 h. The solution is filtered, and the resin is then washed with a small amount of DCM and mixed with the cleavage solution. The resulting solution is poured into 7-10 volumes of chilled (0 °C) methyl tert-butyl ether (MTBE). The suspension is aged at 0 °C for 30 min, then the resulting precipitate is centrifuged and the clear solution is decanted. The residue is suspended in an equal volume of MTBE, and the resulting suspension is again centrifuged and decanted. After decanting, the clear MTBE solution of the precipitated peptide is dried under vacuum at 40 °C overnight.

[0055] Synthesis of Compound 1 by Method 1

[0056] [ka]

[0057] Compound 1 can be prepared substantially by the process described in WO 2020 / 159949.

[0058] Eicosanedioic acid, mono(1,1-dimethylethyl) ester (15.0 kg, limiting reagent), and N-hydroxy-succinimide (1.2 equivalents) are dissolved in ethyl acetate at 27°C. A solution of DCC (1.25 equivalents) is added to the ethyl acetate, and the reaction is stirred at 22°C for 24 hours. The resulting DCU by-product is filtered off, and the organic phase is then extracted three times with 5% aqueous NaCl. After extraction, the organic phase is concentrated and coevaporated with isopropanol, followed by crystallization by adding heptane. After filtration, the filter cake is rinsed with heptane and dried at 25°C to give 17.0 kg of INT1 in 87% yield and 99% purity.

[0059] H-Glu-OtBu (7.7 kg, 1.1 equiv.) is dissolved in DCM (54 L) at 20 °C, then a solution of TMSA (11.3 kg) dissolved in DCM (7 L) is added, and the reaction mixture is stirred at 40 °C for 1 h. INT1 (17.0 kg) is added to the DCM solution at room temperature and stirred for 8 h. After the reaction is complete, DCM is exchanged for ethyl acetate by distillation. The organic phase is washed three times with 2% aqueous KHSO4 / NaCl solution, then four times with 2% aqueous NaCl solution. After separating and removing the aqueous phase, the organic phase is concentrated with isopropanol, diluted with isopropanol, and then crystallized by adding water. After filtration, the filter cake is washed with a water / isopropanol mixture and then dried at 30 °C to produce 17.3 kg of INT2 in 86% yield and 99% purity.

[0060] INT2 (17.3 kg) and N-hydroxy-succinimide (4.1 kg, 1.2 equiv.) are dissolved in ethyl acetate (336 kg) at 27 °C. A solution of DCC (8.33 kg, 1.25 equiv.) is added to the ethyl acetate, and the reaction is stirred at 22 °C for 24 h. The resulting DCU by-product is filtered off. The organic phase is concentrated and coevaporated with isopropanol, followed by crystallization by cooling the isopropanol solution (approximately 125 L). The filter cake is then rinsed with chilled isopropanol and dried at 25 °C to give 16.3 kg of INT3 in 81% yield and 96% purity.

[0061] 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 a solution of TMSA (7.68 kg, 59.9 mol) is added to DCM (6.2 L), and the reaction mixture is then stirred at 40 °C for 1 h. 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 is stirred for 12 h, and upon completion, the mixture is concentrated and then exchanged into ethyl acetate. The organic phase is washed three times with 2% aqueous KHSO4 / NaCl (approximately 200 L), then four times with 2% aqueous NaCl (approximately 200 L) to reach a target pH of 4.5. The organic phase is concentrated and exchanged into acetonitrile. The acetonitrile solution is cooled to -20°C, and the resulting suspension is then aged at -20°C for 15 hours. The mixture is filtered, and the filter cake is rinsed with chilled acetonitrile and then dried below 0°C to give 18.4 kg of Preparation 6 (88% yield) with 96% purity. Overall yield = 53%.

[0062] Synthesis of Compound 1 by Method 2 Alternatively, preparation 6 can be prepared using solid phase peptide synthesis using a peptide synthesizer. Standard coupling procedures are utilized. Standard coupling conditions: Deprotection with 0.133 M, 2.0 equiv. HATU, 5.0 equiv. DIEA, ambient temperature, 3 h, 20% piperidine / DMF 3 x 15 min. Resin Loading: FmocNH-AEEA (0.99 mmol / g) on ​​2-CTC resin: 1.01 g for each parallel reaction. Automated program using DMF swelling followed by Pip / DMF; DMF wash; and amino acid, DIEA, HATU mix; and DMF wash cycle, followed by drying. The resin is cleaved by stirring the combined lots with 30% HFIP / DCM (240 mL) for 1.5 hours. The resin is filtered, washed, and the solvent is removed from the filtrate under vacuum. The resulting oil is dissolved in acetonitrile, and the solvent is again removed. This procedure provides 30.47 g (146% of theoretical yield) of a viscous yellow oil containing 52.3 area % of the desired product by UPLC analysis. The crude product is purified by flash chromatography (500 grams of silica gel, eluting with 85% DCM / 10% methanol / 5% acetic acid, collecting 38 × 100 mL fractions). The previously chromatographed concentrate (17.94 g) is crystallized to give 13.4 g (74.7% yield) with a UPLC purity of 91.65 area %. [Example]

[0063] Example 1 Example (linear SPPS) SEQ ID NO:1 can be prepared substantially by the process described in WO 2020 / 159949, except for using Fmoc-L-Lys(mtt)-OH instead of Fmoc-L-Lys(IVDde)-OH for the addition of lysine at position 20 of SEQ ID NO:1.

[0064] [ka]

[0065] SEQ ID NO:1 (tirzepatide linear 1-39 with Lys-20 mtt protection on resin) (2.0 grams) was suspended in 15 mL of 30% (v / v) HFIP / DCM in a 45 mL Symphony X reactor. The reaction was mixed at ambient temperature for 1 hour and then drained. The resin was again treated with 15 mL of 30% HFIP / DCM, mixed for 1 hour, and drained. The resin was washed five times with 15 mL portions of DCM and then vacuum dried at 35° C. to a constant weight to provide SEQ ID NO:2.

[0066] [ka]

[0067] SEQ ID NO:2 (tirzepatide linear 1-39 with Lys-20 NH2 on Sieber resin) (2.0 g) was suspended in 20 mL of 5% (v / v) TFA / DCM and mixed at ambient temperature for 30 minutes. The resin was removed by filtration through a fritted filter and rinsed with 10 mL of DCM. The combined filtrate and washes were treated with 1.05 mL (1 equivalent based on the TFA used) of pyridine to neutralize the TFA. The DCM was removed under reduced pressure, and the residue was dissolved in 4 mL of DMF. This solution was added in portions to 20 mL of cold water to precipitate the cleaved peptide. After cooling at 5°C for approximately 30 minutes, the solid was filtered, washed with 20 mL of water, and dried under vacuum at 45°C to a constant weight (1.25 g) to obtain SEQ ID NO:3.

[0068] [ka]

[0069] To a solution of SEQ ID NO:3 (tirzepatide 1-39 Lys-20 NH2 linear peptide) (500 mg, 0.091 mmol), compound 1 (2.0 equivalents), and 141.3 mg (2.0 equivalents) of PyBOP in 5 mL of DMF was added DIEA (48 μL, 3.0 equivalents). The reaction solution was stirred at ambient temperature for 18 hours. Analysis by HPLC showed complete conversion of the linear peptide to the desired product. The solution was added in portions to 40 mL of cold water, causing a precipitate to form. After cooling at 5°C for 30 minutes, the solid was collected by filtration on a fritted filter, washed with 20 mL of water, and dried under vacuum to give 535 mg of protected tirzepatide (SEQ ID NO:4).

[0070] [ka]

[0071] The protected tirzepatide (SEQ ID NO: 4) can be deprotected to provide tirzepatide (SEQ ID NO: 5) by a process essentially as described in WO 2020 / 159949.

[0072] [ka]

[0073] Alternatively, mtt is removed from SEQ ID NO:1 to form SEQ ID NO:2, and then Compound 1 is coupled to SEQ ID NO:2 to form SEQ ID NO:6.

[0074] [ka]

[0075] The resin is then removed from SEQ ID NO:6 to form SEQ ID NO:4, which is globally deprotected to form tirzepatide (SEQ ID NO:5).

[0076] Example 2 MTT Process-2 Removal of the mtt group from Lys-20 on the TZP1-39 linear peptide: TZP linear 1-39 (2.0 grams) bearing Lys-20 mtt protection on the resin was suspended in 15 mL of 30% (v / v) HFIP / DCM in a 45 mL Symphony X reactor. The reaction was mixed at ambient temperature for 1 hour and then drained. The resin was again treated with 15 mL of 30% HFIP / DCM, mixed for 1 hour, and drained. The resin was washed five times with 15 mL portions of DCM and then vacuum dried at 35° C. to a constant weight.

[0077] [ka]

[0078] TZP1-39 Lys-20 NH from Sieber resin 2 Soft cleavage of linear peptides: TZP linear 1-39 (2.0 g) bearing Lys-20 NH2 on Sieber resin was suspended in 20 mL of 5% (v / v) TFA / DCM and mixed at ambient temperature for 30 min. The resin was removed by filtration through a fritted filter and rinsed with 10 mL of DCM. The combined filtrate and washes were treated with 1.05 mL (1 equivalent based on the TFA used) of pyridine to neutralize the TFA. The DCM was removed under reduced pressure, and the residue was dissolved in 4 mL of DMF. This solution was added in portions to 20 mL of cold water to precipitate the cleaved peptide. After cooling at 5 °C for approximately 30 min, the solid was filtered, washed with 20 mL of water, and dried under vacuum at 45 °C to a constant weight (1.25 g).

[0079] [ka]

[0080] [ka]

[0081] Coupling of TZP1-39 Lys-20 NH2 linear peptide with (S)-22-(tert-butoxycarbonyl)-45,45-dimethyl-10,19,24,43-tetraoxo-3,6,12,15,44-pentaoxa-9,18,23-triazahexatetracontanoic acid: To a solution of TZP1-39 Lys-20 NH2 linear peptide (500 mg, 0.091 mmol), (S)-22-(tert-butoxycarbonyl)-45,45-dimethyl-10,19,24,43-tetraoxo-3,6,12,15,44-pentaoxa-9,18,23-triazahexatetracontanoic acid (2.0 equiv.), and 141.3 mg (2.0 equiv.) of PyBOP in 5 mL of DMF was added DIEA (48 μL, 3.0 equiv.). The reaction solution was stirred at ambient temperature for 18 h. Analysis by HPLC indicated complete conversion of the linear peptide to the desired product. The solution was added in portions to 40 mL of cold water, resulting in the formation of a precipitate. After cooling at 5° C. for 30 minutes, the solid was collected by filtration on a fritted filter, washed with 20 mL of water, and dried in vacuo to give 535 mg of protected tirzepatide (SEQ ID NO: 4).

[0082] The protected tirzepatide (SEQ ID NO: 4) can be deprotected to provide tirzepatide (SEQ ID NO: 5) by a process essentially as described in WO 2020 / 159949.

[0083] Chiral purity determination The structures of tirzepatide fragments, including amino acid sequences (residue numbers represent positions in the fully assembled tirzepatide drug substance), are prepared substantially as described herein. L- and D-isomer fragment materials were synthesized as high-purity amorphous solids by Eli Lilly and Company (Indianapolis, USA). Fragment samples were fully characterized for identity, purity, and content. Tirzepatide fragments are "protected" peptides in which the active side chains of amino acids (e.g., hydroxyl, carboxyl, amino) are protected with functional groups such as Fmoc, trityl (Trt), tert-butyloxycarbonyl (Boc), and tertiary butyl (tBu). Chiral purity analysis of the L-isomer fraction material was also performed using established GCMS and LCMS methodologies (Kalikova et al., Separations 3 pp. 30-48 (2016); Liardon et al., J. Chromatogr. A 203 pp. 385-395 (1981)).

[0084] array SEQ ID NO:5 Tirzepatide YX1EGTFTSDYSIX2LDKIAQKAFVQWLIAGGPSSGAPPPS X1 is Aib, X2 is Aib, and K at position 20 is (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γGlu)1-CO—(CH2) 18 It is chemically modified through conjugation of the K side chain with -CO2H to the epsilon-amino group, and the C-terminal amino acid is amidated as a C-terminal primary amide.

Claims

1. A compound of SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof.

2. 2. The compound of claim 1, wherein the compound is: 【Chemistry 1】

3. 1. A process for making tirzepatide (SEQ ID NO: 5), comprising: a. deprotecting the compound of SEQ ID NO: 1; b. Removing the resin; c. Coupling SEQ ID NO:3 with Compound 1; d. deprotecting SEQ ID NO:

4.

4. Deprotecting the compound of SEQ ID NO: 1 to obtain a compound of (SEQ ID NO: 2), The resin was removed to give (SEQ ID NO: 3), 4. The process of claim 3, wherein SEQ ID NO: 3 is coupled with Compound 1 to obtain (SEQ ID NO: 4).

5. 1. A process for making tirzepatide (SEQ ID NO: 5), comprising: a. Deprotecting the compound of SEQ ID NO: 1 (SEQ ID NO: 2); b. Removing the resin to obtain (SEQ ID NO: 3); c. coupling SEQ ID NO:3 with Compound 1 to obtain (SEQ ID NO:4); d. deprotecting SEQ ID NO:4 to obtain tirzepatide (SEQ ID NO:5).

6. 1. A continuous flow process for preparing tirzepatide (SEQ ID NO: 5), comprising: a. deprotecting the compound of SEQ ID NO: 1; b. Coupling SEQ ID NO:2 with Compound 1; c. A continuous flow process including deprotection and resin removal, using nanofiltration in-line to provide purification and solvent exchange.

7. A continuous flow process as described in c, wherein chromatography is used in-line.

8. 1. A continuous flow process for making tirzepatide (SEQ ID NO: 5), comprising: a. deprotecting the compound of SEQ ID NO: 1; b. Removing the resin and optionally arranging chromatography and nanofiltration in-line; c. coupling SEQ ID NO:3 with Compound 1 and placing nanofiltration in-line; d. Deprotecting SEQ ID NO:4 by in-line chromatography and nanofiltration.

Images