Method for preparing GIP / GLP1 dual agonists

Abstract

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

Description

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

[0002] Diabetes is a chronic disease characterized by hyperglycemia due to defects in insulin secretion, insulin action, or both. In type 2 diabetes ("T2D"), the combination of impaired insulin secretion and insulin resistance leads to elevated blood glucose levels. Telportide, a dual GIP / GLP1 agonist, is described and claimed in U.S. Patent 9,474,780 (the "'780 patent"). Telportide can be used to treat T2D.

[0003] US9474780 summarizes peptides and methods for preparing GIP / GLP1 dual agonists. WO2020 / 159949 describes methods for preparing tilportide; however, linear process improvements are needed to facilitate large-scale commercial production.

[0004] There is a need for methods and intermediates to achieve improved technologies for producing tilpotide that combine the advantages of achieving commercially desirable purity and efficiency. Similarly, there is a need for efficient and environmentally friendly "green" methods that include stable intermediates and require fewer purification steps to produce tilpotide. There is also a need for improved technologies that provide methods for producing tilpotide that minimize waste streams, thereby improving environmental and operator safety. The large-scale, pharmaceutically desirable production of tilpotide faces numerous technical challenges that may affect overall yield and purity. There is a need for methods that avoid the use of transition metals and / or harsh reaction conditions that are incompatible with peptide synthesis.

[0005] The present invention aims to address these needs by providing novel intermediates and methods for preparing tilpotide (SEQ ID NO: 5) or a pharmaceutically acceptable salt thereof. The improved methods for preparing tilpotide of the present invention provide intermediates and process reactions that represent combinatorial advances, including an efficient, fewer-step route while maintaining high quality and purity. Importantly, the improved methods and intermediates reduce resource intensity and minimize waste streams.

[0006] The improved methods described herein provide various embodiments of intermediates for the production of telpotide.

[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 method for preparing tulpotilide, comprising preparing a protected compound of SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof. The present invention provides a method for preparing tulpotilide, comprising deprotecting a compound of SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof. The present invention provides a method for preparing tulpotilide, comprising cleaving a compound of SEQ ID NO: 2 or a pharmaceutically acceptable salt thereof from a resin. The present invention provides a method for preparing tulpotilide, comprising deprotecting a compound of SEQ ID NO: 3 or a pharmaceutically acceptable salt thereof. The present invention provides a method for preparing tulpotilide (SEQ ID NO: 5), comprising acylating lysine to form a compound of SEQ ID NO: 4 or a pharmaceutically acceptable salt thereof, followed by deprotection to form tulpotilide.

[0009] The present invention provides a method for preparing a resin-bound protected peptide of SEQ ID NO: 1 in which the epsilon amino group of the lysine amino acid is protected with MTT. The present invention also provides a method for selectively removing the MTT protection from the epsilon amino group of the lysine amino acid in the resin-bound protected peptide of SEQ ID NO: 1 to form a protected peptide of SEQ ID NO: 2. The present invention also provides a method for selectively cleaving the resin-bound protected peptide of SEQ ID NO: 2 to form a compound of SEQ ID NO: 3. The present invention also provides a method for acylation of the unprotected epsilon amino group of lysine of SEQ ID NO: 3 with Compound 1 to form a compound of SEQ ID NO: 4.

[0010] The present invention provides a method for determining the chiral purity of SPPS prepared peptides by using a chiral high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS / MS) method.

[0011] In one embodiment, the method for preparing telpotide (SEQ ID NO: 5) comprises

[0012] a) deprotecting the compound of SEQ ID NO: 1 to obtain the compound of SEQ ID NO: 2;

[0013] b) removing the resin to obtain the compound of SEQ ID NO: 3;

[0014] c) coupling SEQ ID NO: 3 with compound 1 to obtain SEQ ID NO: 4;

[0015] d) deprotecting SEQ ID NO: 4 to obtain telpotide (SEQ ID NO: 5).

[0016] In one embodiment, the method for preparing telpotide (SEQ ID NO: 5) comprises

[0017] a) deprotecting the compound of SEQ ID NO: 1 to obtain the compound of SEQ ID NO: 2;

[0018] b) coupling SEQ ID NO: 2 with compound 1 to obtain SEQ ID NO: 6;

[0019] c) deprotecting and removing the resin to obtain telpotide (SEQ ID NO: 5);

[0020] d) removing SEQ ID NO: 6 from the resin to obtain SEQ ID NO: 4; and

[0021] e) deprotecting SEQ ID NO: 4 to obtain telpotide (SEQ ID NO: 5).

[0022] In one embodiment, the method for preparing telpotide (SEQ ID NO: 5) comprises

[0023] a) deprotecting the compound of SEQ ID NO: 1 to obtain the compound of SEQ ID NO: 2;

[0024] b) coupling SEQ ID NO: 2 with compound 1 to obtain SEQ ID NO: 6;

[0025] c) removing SEQ ID NO: 6 from the resin to obtain SEQ ID NO: 4; and

[0026] d) deprotecting SEQ ID NO: 4 to obtain telpotide (SEQ ID NO: 5).

[0027] In one embodiment, a continuous flow method for preparing telpotide (SEQ ID NO: 5) is provided, comprising:

[0028] a) deprotecting the compound of SEQ ID NO: 1 to obtain the compound of SEQ ID NO: 2;

[0029] b) removing the resin to obtain a compound of SEQ ID NO: 3; wherein nanofiltration is optionally placed in line to purify and provide solvent exchange;

[0030] c) coupling SEQ ID NO: 3 with compound 1 to obtain SEQ ID NO: 4; wherein nanofiltration is placed online to purify and provide solvent exchange;

[0031] d) Deprotection of SEQ ID NO: 4 to obtain telpotide (SEQ ID NO: 5), wherein nanofiltration was placed in-line for purification and to provide solvent exchange.

[0032] In one embodiment, a continuous flow method for preparing telpol peptide (SEQ ID NO: 5) is provided, comprising:

[0033] a) deprotecting the compound of SEQ ID NO: 1 to obtain the compound of SEQ ID NO: 2;

[0034] b) coupling SEQ ID NO: 2 with compound 1 to obtain SEQ ID NO: 6;

[0035] c) removing SEQ ID NO: 6 from the resin to obtain SEQ ID NO: 4; and

[0036] d) Deprotection of SEQ ID NO: 4 to obtain telpotide (SEQ ID NO: 5), wherein nanofiltration was placed in-line for purification and to provide solvent exchange.

[0037] In one embodiment, to improve efficiency, the continuous flow method for preparing telpotide (SEQ ID NO: 5) and chromatographic purification comprises:

[0038] a) deprotecting the compound of SEQ ID NO: 1 to obtain the compound of SEQ ID NO: 2;

[0039] b) removing the resin to obtain a compound of SEQ ID NO: 3; wherein chromatography and nanofiltration are optionally placed online to purify and provide solvent exchange;

[0040] c) coupling SEQ ID NO: 3 with compound 1 to obtain SEQ ID NO: 4; wherein nanofiltration is placed online to purify and provide solvent exchange;

[0041] d) Deprotection of SEQ ID NO: 4 to obtain Telpol peptide (SEQ ID NO: 5), which was subjected to online chromatography nanofiltration for purification and to provide solvent exchange.

[0042] In one embodiment, a continuous flow method and chromatographic purification for preparing tilpoxetine (SEQ ID NO: 5) is provided, comprising:

[0043] a) deprotecting the compound of SEQ ID NO: 1 to obtain the compound of SEQ ID NO: 2;

[0044] b) removing the resin to obtain a compound of SEQ ID NO: 3; wherein chromatography and nanofiltration are placed online to purify and provide solvent exchange;

[0045] c) coupling SEQ ID NO: 3 with compound 1 to obtain SEQ ID NO: 4; wherein nanofiltration is placed online to purify and provide solvent exchange;

[0046] d) Deprotection of SEQ ID NO: 4 to obtain Telpol peptide (SEQ ID NO: 5), which was subjected to online chromatography nanofiltration for purification and to provide solvent exchange.

[0047] Provided are methods for selectively acylating unprotected lysine amino acids. Provided are methods for selectively acylating lysine amino acids in peptides, comprising coupling resin-bound peptide-lysine-NH2 with tert-butyl-eicosanedioyl-Glu-(O-tert-butyl)-(8-amino-3,6-dioxaoctanoic acid)-(8-amino-3,6-dioxaoctanoic acid)-OH. Provided are methods for preparing telpotide, comprising deprotecting a compound of SEQ ID NO: 4 or a pharmaceutically acceptable salt thereof.

[0048] The present invention provides a method for preparing tulpoic acid, comprising cleaving a compound of SEQ ID NO: 2 or a pharmaceutically acceptable salt thereof from a resin. The present invention provides a method for preparing tulpoic acid, comprising deprotecting a compound of SEQ ID NO: 3 or a pharmaceutically acceptable salt thereof. The present invention provides a method for preparing tulpoic acid, comprising acylating lysine to form a compound of SEQ ID NO: 4 or a pharmaceutically acceptable salt thereof.

[0049] A method for selectively acylating a lysine amino acid is provided, wherein the resin-bound peptide-lysine-NH2 is a compound of SEQ ID NO: 2:

[0050] or a pharmaceutically acceptable salt thereof.

[0051] The improved method uses an intermediate protected by MTT on lysine, which has an orthogonal protection purity higher than that of similar intermediates used in known methods. In addition, when using the MTT-protected intermediate to synthesize tirpotide on resin, orthogonal deprotection is performed at the lysine 20 position to remove the MTT and couple it with (S)-22-(tert-butyloxycarbonyl)-45,45-dimethyl-10,19,24,43-tetraoxo-3,6,12,15,44-pentaoxa-9,18,23-triazatetradecanoic acid to obtain the desired purity. In addition, SEQ ID NO: 6 can be cleaved from the resin to form SEQ ID NO: 4, which is then deprotected to obtain tirpotide, thereby achieving continuous flow synthesis.

[0052] Both methods for synthesizing tilpoxetine using MTT provide improved purity and yield compared to known linear methods. Furthermore, due to the LPPS conditions, another advantage of the methods described herein is the ability to reduce the stoichiometry of (S)-22-(tert-butyloxycarbonyl)-45,45-dimethyl-10,19,24,43-tetraoxo-3,6,12,15,44-pentaoxa-9,18,23-triazatetrahexadecanoic acid to approximately 1 equivalent and potentially amenable to flow synthesis methods. Reducing the need for excess (S)-22-(tert-butyloxycarbonyl)-45,45-dimethyl-10,19,24,43-tetraoxo-3,6,12,15,44-pentaoxa-9,18,23-triazatetrahexadecanoic acid has environmental and commercial significance for large-scale peptide synthesis.

[0053] Determination of chiral purity is crucial for assessing the quality of peptide pharmaceutical products. For synthetic peptides, unwanted D-isomers can be introduced as impurities in the amino acid starting materials, formed during peptide synthesis, and sometimes even during the product's shelf life. This paper describes a chiral high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS / MS) method that facilitates rapid and accurate determination of the chiral purity of amino acids in peptides. The peptides are hydrolyzed in deuterated acid to correct for any racemization that occurs during sample preparation steps. The amino acids are then separated and quantified by chiral chromatography coupled to ESI-MS / MS. Direct chromatographic separation and extraction of selected ion responses after hydrolysis of the amino acid samples for analysis offer efficiency and simplicity by avoiding the derivatization step required by traditional methods. Validation of this method for all 19 chiral proteinaceous amino acids is described, and its practical application in the analysis of a model peptide is demonstrated. The method has been shown to quantify trace levels of D-isomer impurities within the target range of 0.1%–1.0%.

[0054] As used herein, the following abbreviations have the meanings set forth herein: “SPPS” means solid phase peptide synthesis, “MTT” and / or “mtt” means 4-methyltrityl, “Fmoc” means fluorenylmethoxycarbonyl chloride, “Pip” means piperidine, “DIC” means diisopropylcarbodiimide, “Oxyma” means ethyl cyanohydroxyiminoacetate, “DCM” means dichloromethane, “IPA” means isopropyl alcohol, “MTBE” means methyl tert-butyl ether, “TFA” means trifluoroacetic acid, “TIPS” means triisopropylsilane, “DTT” means dithiothreitol, “UPLC” means ultra performance liquid chromatography, “HFIP” means hexafluoroisopropanol, “CTC” means chlorotrityl, and “HATU” means tetrafluoroethylene. (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, "TFET" denotes 2,2,2-trifluoroethanethiol, "DIEA" denotes N,N-diisopropylethylamine, "AEEA" denotes 17-amino-10-oxo-3,6,12,15-tetraoxa-9-azaheptadecanoic acid, "TCEP" denotes tris(2-carboxyethyl)phosphine, "DCU" denotes dicyclohexylurea, "DCC" denotes dicyclohexylcarbodiimide, "TMSA" denotes trimethylsilylamine, "HOBt" denotes hydroxybenzotriazole, "HRMS" denotes high-resolution mass spectrometry, and "LPPS" denotes liquid chromatography-mass spectrometry. Phase peptide synthesis, "MSMPR" stands for mixed product mixing suspension reactor, "MPA" stands for mobile phase A, "MPB" stands for mobile phase B, "L-GSH" stands for L-glutathione reducing solution, "TZP" stands for telportide, "AP" stands for active pharmaceutical ingredient, "API" stands for active pharmaceutical ingredient, "PyBOP" stands for (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, "DEA" stands for diethylamine, "TBTU" stands for 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylammonium tetrafluoroborate, "TNTU" stands for 2-(5-norbornene-2,3-dicarboximide)-1,1,3,3-tetramethyluronium tetrafluoroborate, and "PyOxim" stands for 1-Cyano-2-ethoxy-2-oxoethyleneaminooxy-tripyrrolidinylphosphonium hexafluorophosphate, "PyClock" stands for 6-chloro-benzotriazol-1-yloxy-tripyrrolidinylphosphonium hexafluorophosphate. As described herein, the single-letter amino acid abbreviations are shown in bold, while atoms are shown in non-bold text, often in a smaller font, to distinguish them from the single-letter amino acid abbreviations. As used herein, when an amino acid abbreviation appears with a number above it, the number refers to the corresponding amino acid position in the final telportipeptide product. These numbers are provided for convenience, and the presence or absence of these numbers in the sequence does not affect the amino acid sequence or the peptide shown in the sequence. As used herein, the term "protected" refers to the presence of a protecting group at the designated position.Those skilled in the art will recognize that various protecting groups are well-known, and alternative protecting groups may be suitable for ad hoc methods. As used herein, "online" refers to continuous flow synthesis, wherein the features are provided during building-up so that building-up can continue uninterruptedly. In one embodiment, online nanofiltration can be used for purification and / or solvent exchange according to synthesis needs. In one embodiment, online chromatography can be used online during building-up.

[0055] Those skilled in the art will appreciate that alternative resins exist for constructing the peptides described herein. For example, Sieber and Rink amide resins are well known to those skilled in the art for use in preparing the peptides disclosed herein; however, alternative resins may also be selected for use in preparing the peptides described herein. For example, but not limited to, 2-CTC and related resins may be used to prepare the target peptide, followed by a C-terminal amidation step.

[0056] The methods described herein are suitable for telescoped steps, i.e., multiple method steps are run simultaneously, encompassing multiple unit operations, thereby enabling the assembly of multiple preassembled peptide units through continuous unit operations, or "continuous flow." Continuous flow methods are enabled by fume hood designs that support continuous flow. Continuous flow synthesis allows for safe, cost-effective, and efficient continuous production to meet commercial peptide demand.

[0057] The methods provided herein can use nanofiltration for purification and solvent exchange to eliminate the need for additional separation steps and enable a continuous processing platform.

[0058] A continuous production module (skid) based on unit operations, housed in a fume hood, can be used to perform peptide unit coupling reactions in a flow process. Precisely control the feed to the peptide unit coupling reaction to achieve high reaction conversions while maintaining tight stoichiometric control, unaffected by the expected variability from solid-phase synthesis fragments. The feed flow rate for the continuous flow method can be automatically updated based on method needs.

[0059] Nanofiltration can be used for purification and solvent exchange of coupled peptide solutions. Nanofiltration can eliminate the need for separation of amorphous solids and enable a fully telescoped process of reactions through peptide units. A standby nanofiltration skid allows continued production during scheduled nanofiltration maintenance.

[0060] The peptide solution can be precipitated and separated in a dual train setup, also known as batch precipitation and separation. After crude peptide assembly is complete, the resulting peptide solution can be precipitated and separated using batch precipitation and separation equipment. The dual train setup allows for continuous flow reactions, along with batch precipitation and separation unit operations. Pack-offering canister packaging technology can be used to protect operators and equipment.

[0061] Equipment used in the synthetic peptide production process can be equipped with clean-in-place circuits and cleaning batch recipes to ensure convenient, efficient and effective cleaning.

[0062] Solid-phase synthesis allows for the isolation of growing peptide fragments, reducing the need for isolation of amorphous intermediates and the potential need for byproduct removal and washing steps to achieve continuous construction. Traditional liquid-phase coupling often requires complex separation steps to remove byproducts. To eliminate the complexity of intermediate isolation between couplings of individual units, washing of the coupled peptide in solution can be used.

[0063] Nanofiltration can provide concentration, washing, and solvent exchange following the coupling method. Because peptide solubilization requires challenging polar aprotic solvents, non-traditional ceramic membranes can provide a useful alternative to more typical polymeric membranes.

[0064] Nanofiltration offers an efficient, scalable, and economical method for integrating multiple sequential coupling steps into complete peptide assembly systems. Nanofiltration technology enables the use of continuous synthesis platforms by eliminating time-consuming intermediate isolation steps. Nanofiltration is effective for peptides with a wide range of molecular weights, making this platform applicable to multiple peptide projects.

[0065] Ceramic membranes are made of inorganic materials such as, but not limited to, aluminum oxide, zirconium oxide, and silicon carbide. The inorganic materials used to make ceramic membranes enable them to be used with corrosive media such as acids and strong solvents, in which polymer membranes may fail, and therefore the ceramic membranes are suitable for the liquid phase coupling reactions described herein. Ceramic membranes can be made into tubular cross-flow configurations, which are suitable for the continuous coupling steps described herein. Ceramic membranes are available from a number of suppliers, including but not limited to: Altech (Gladbeck, Germany); CoorsTek (Golden, Colorado); Inopor (Scheβlitz, Germany); Mantec Technical Ceramics Ltd (Staffordshire, England); and OrelisEnvironnement / Alsys group (Salindres, France).

[0066] The underivatized amino acid method can be used with a stationary phase to optimize chiral purity determination by quantifying trace levels of D-isomers in synthetic peptides. Samples are hydrolyzed in deuterium chloride (DCl) to allow for the exclusion of amino acids racemized during sample preparation by mass spectrometry detection. Eliminating the sample derivatization step from the method can help simplify the method and potentially increase throughput. The data presented demonstrate chiral HPLC-ESI-MS / MS analysis of a mixture of all 19 chiral D / L natural amino acid pairs, with quantification of the D-isomers by monitoring the extracted ion chromatograms (EICs) associated with peptide ions unique to each amino acid. The feasibility of this method for the quantitative determination of trace levels of D-isomers in a matrix containing L-isomers is demonstrated by measuring linearity, accuracy, precision, and detection / quantification limits. This supports a synthetic peptide manufacturing process control limit of 0.5% and a method reporting limit of 0.1% for each D-isomer, meeting pharmaceutical industry regulatory guidelines for synthetic peptides. The chiral HPLC-ESI-MS / MS method was validated by the analysis of four model peptides (each consisting of 8-14 amino acid residues).

[0067] The four model peptides used in this study each consist of 8-14 amino acid residues and are described in Table 1 by amino acid content and residue number. Both L- and D-isomer peptide materials were synthesized as high-purity amorphous solids. The peptides were fully characterized for their properties, purity, and content. The samples were synthesized as "protected" peptides, in which the reactive side chains of the amino acids (e.g., hydroxyl, carboxyl, amino, etc.) were protected with functional groups such as Fmoc, trityl (Trt), tert-butyloxycarbonyl (Boc), and tert-butyl (tBu), as shown in Table 1. The chiral purity of the peptide samples was also compared using conventional GCMS and LCMS methods.

[0068] Table 1: Amino acid content of model peptide samples

[0069]

[0070] *Aib (2-aminoisobutyric acid) is an achiral, non-proteinogenic amino acid

[0071] Fragment A

[0072]

[0073] Fragment B

[0074]

[0075] Fragment C

[0076]

[0077] Fragment D

[0078]

[0079] Sample preparation

[0080] The nominal target concentration for each amino acid was 0.1 mg / mL. With the exception of aspartic acid, cysteine, and tyrosine, all amino acid standards were prepared in water. These three amino acids required approximately 4% formic acid to dissolve (dissolve the three amino acids in formic acid and then dilute to volume with water). Amino acid standard solutions were prepared at concentrations of approximately 0.0005 mg / mL to 0.002 mg / mL to represent a nominal amino acid concentration range of 0.05–2.0% in the peptide hydrolyzate mixture.

[0081] To analyze Fmoc-protected peptide fragments B and C, samples were incubated in a piperidine solution (3 mL piperidine + 10 mL dichloromethane) at a concentration of approximately 1 mg / mL (providing an amino acid concentration of at least 0.1 mg / mL for a 10-amino acid peptide) for 15 min at 2-8°C to remove the Fmoc protecting group, followed by hydrolysis. After Fmoc removal, the samples were dried under a stream of nitrogen for 4 hours. All dried peptide samples were digested by acid hydrolysis in 1.0 mL of 5N DC1 at a concentration of approximately 1 mg / mL in 10 mL glass vials with Teflon heat-resistant caps at 110°C for 4 hours. To prevent oxidation of tryptophan to N'-formylkynurenine or related substances, a sample of peptide fragment B was mixed with 5N DC1 + 1% phenol, purged with nitrogen, and carefully sealed with Teflon tape and parafilm prior to hydrolysis at 110°C. In addition to cleaving the peptide into its individual amino acid components, the remaining protecting groups (tBu, Boc, and Trt) are also removed during the hydrolysis step. After hydrolysis, the sample is cooled to ambient temperature and 2 mL of water is added. The sample is then eluted using a Genevac TM The samples were dried for 12 h using an EZ-2 Elite HCl compatible centrifugal vacuum concentrator (Genevac Ltd. Ipswitch, UK) and the acid was removed by lyophilization. As a final step, the samples were reconstituted with water to the original volume of 1 mL and then analyzed by chiral HPLC-ESI-MS / MS. The proline sample was analyzed using a ZWIX(-) column and further diluted 1:1 with water before injection.

[0082] Chiral HPLC-ESI-MS / MS

[0083] The UPLC system consists of a binary solvent manager and an autosampler. The chromatography was performed on a CR-I(+)150x3.0mm ID 5μm column (Chiral Technologies, Inc. West Chester, PA) with isocratic elution and a mobile phase composition of 5% A (0.5% TFA in water) and 95% B (85% ACN / 15EtOH / 0.5TFA; v / v / v). The flow rate was 0.4mL / min, the injection volume was 1μL, and the column temperature was 30°C. A single Separation was performed on a ZWIX(-)150x3.0mm ID 5μm column (Chiral Technologies, Inc. West Chester, PA) using an isocratic mobile phase consisting of 25mM formic acid, 25mM ammonium formate in methanol / water (98 / 2 v / v).

[0084] The UPLC was coupled to a mass spectrometer equipped with a Turbo Spray IonDrive and operated in multiple reaction monitoring (MRM) mode. Separation was performed using the chromatographic conditions described above, with a diverter valve set to divert the first two minutes of sample to waste. The mass spectrometer was operated in positive ion polarity with an ion spray voltage set to 5500 V and a temperature set to 550°C. The MRM transitions for each amino acid can be found in Table 2, along with their respective Q1 and Q3 m / z values ​​(all set to unit resolution), as well as optimized values ​​for the declustering potential, entrance potential, collision energy, and collision cell exit potential. The transition from 132.1 to 86.0 m / z represents Leu and Ile, as both amino acids behave in this manner, but this transition does not provide any specificity between this pair of isobaric amino acids. However, this chromatography method effectively separates the Leu and Ile isomers (20, 25). Furthermore, the transition from 132.1 to 69.0 m / z is specific for isoleucine and provides a transition useful for isoleucine quantification. Other relevant instrument parameters include: curtain gas 20 PSI, ion source gas 1 40 PSI, ion source gas 2 60 PSI, and collision gas setting low. Data acquisition and processing were performed using Analyst software (SCIEX).

[0085] Table 2: List of target amino acids and their related MRM conditions

[0086]

[0087] Chiral HPLC Separation of Amino Acids

[0088] Both crown ether and quinine / quinidine-derived zwitterionic stationary phases used in hydrophilic interaction chromatography (HILIC) mode have been demonstrated to be highly effective chiral selectors for the separation of amino acid enantiomers. The crown ether of the stationary phase interacts strongly with the amino groups protonated by the strongly acidic TFA mobile phase, and then the binaphthyl moiety favors stereoselective 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, which has a poor affinity for the amino groups of secondary amines. The chiral separation of proline is performed by ZWIX offers this alkaloid-derived zwitterionic stationary phase, which is believed to induce selectivity through ion pairing in mobile phases containing acidic and basic additives. Both packings offer the ability to separate isomeric analytes in an order that can be tuned by selecting the appropriate chiral configuration of the stationary phase. CR-I(+) and ZWIX(-) packings are preferred because they promote the elution of the D-isomer before the L-isomer, thereby minimizing interference caused by tailing of the high-concentration L-isomer peak.

[0089] use CR-I(+) and The chiral chromatography method for the ZWIX(-) column was designed by referring to the parameters for the analysis of D-amino acids in foods described previously. However, in the present work, the two CR-I(+) columns were connected in series to provide optimal resolution. To determine the D-isomer impurities in the synthetic peptides, the chromatographic separation was combined with a high-low sample concentration chromatography strategy (26). The high-low chromatography method is named because a pair of sample solutions is used, with "high" corresponding to a nominal sample concentration of approximately 0.1 mg / mL for each L-amino acid in the hydrolysis solution mixture and "low" indicating 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, where the peak area of ​​the L-isomer in the low concentration sample is effectively used as an external standard calibrator for the area quantification of the % D-isomer peak area measured in the high concentration sample:

[0090]

[0091] Here, area D represents the peak area of ​​the D-isomer in a 0.1 mg / mL sample, and area L represents the peak area of ​​the L-isomer in a 0.001 mg / mL sample. This strategy offers two significant advantages: increased sensitivity, which helps lower detection limits, and simplified quantitative calibration. The high / low strategy eliminates the need for a set of 19 external standards for each amino acid that would otherwise be used.

[0092] Chiral HPLC-ESI-MS / MS Analysis of Amino Acids

[0093] In chiral HPLC-ESI-MS / MS analysis, protonated molecular ions ([M+H]+) are selected as precursor ions for all target compounds based on the expected m / z values ​​corresponding to each non-racemic (i.e., non-deuterated) amino acid in DCl. Peptidation is induced, and the ion signal response of the product ion unique to each amino acid is measured. Table 5 lists the precursor and product ions used for quantification of each amino acid. After sample preparation and analysis, the extracted ion chromatogram (EIC) associated with the product ion response associated with each amino acid will represent the D- and L-amino acids inherent in the peptide prior to hydrolysis. As mentioned in the introduction, any racemic amino acids present during hydrolysis are excluded from the precursor ions because the incorporation of deuterium increases the molecular weight by 1 atomic mass unit. Using this approach, all amino acids present in the peptide can be analyzed post-hydrolysis in a single chiral HPLC-ESI-MS / MS experiment by monitoring the EIC corresponding to the expected product ions eluting at a defined chromatographic retention time.

[0094] To ensure sensitivity, linearity, and accuracy within the method reporting limit of 0.1% and the quantification range of 0.1%-1.0% for the D-isomer, a set of standards at six concentrations (0.05%, 0.1%, 0.2%, 0.5%, 1.0%, and 2.0%) was prepared and analyzed for each amino acid. Detector response demonstrated excellent sensitivity for all amino acids, with responses at 0.1% yielding S / N > 10 and linearity R 2 =0.9900 (see Table 3). D-isomer spiked samples were also prepared into the corresponding L-isomer matrices. The spiked samples were added to the corresponding L-isomer matrices at levels of 0.1%, 0.2%, 0.5%, and 1.0% with a nominal concentration of 0.1 mg / mL. The method was demonstrated to be capable of measuring the response of the D-isomer with a recovery accuracy of approximately 80-120%, where the % recovery is based on the weighed spiked sample preparation and the spiked response is compared directly to the corresponding standard curve. Table 3 lists the sensitivity, linearity, and accuracy data for all 19 chiral amino acids, and the results support the quantitative capability of the method.

[0095] Table 3: Chiral HPLC-ESI-MS / MS sensitivity, linearity, and accuracy data representing quantification of the D-isomers of all 19 natural chiral amino acids with a support range of 0.10%-1.0%.

[0096]

[0097]

[0098] Batches of all four model peptides were analyzed and reported using both the chiral HPLC-ESI-MS / MS method and the conventional method for comparison and confirmation. Peptides A and D were tested for comparison by derivatization GC-MS, while peptides B and C were tested for comparison by derivatization HPLC-MS. The results, listed in Table 4, show the expected low levels of the D-isomer and good agreement between the new and conventional techniques within the inherent variability estimated based on the accuracy and precision of each method (approximately + / - 0.1%, based on 3x SD of the current method within the range of 0.1-0.2% D-isomer and validation of the GC-MS method (30)).

[0099] Table 4

[0100]

[0101]

[0102] 1Peptides A and D were tested essentially according to the method described by R. Liardon, S. Ledermann and U. Ott, Determination of D-amino acids by deuterium labelling and selected ion monitoring, J. Chromatogr. A 203 (1981) 385-395.

[0103] 2 The testing of peptides B and C was performed essentially according to the method described by K. Kalikova, T. Slechtova and E. Tesarova in Enantiomeric ratio of amino acids as a tool for determination of aging and disease diagnostics by chromatographic measurement, Separations 3 (2016) 30-48).

[0104] Solid-phase peptide synthesis (SPPS) construction was accomplished using standard fluorenylmethoxycarbonyl chloride (Fmoc) peptide chemistry techniques, using an automated peptide synthesizer for sequential couplings. The resin was swollen with DMF and then deprotected using 20% ​​piperidine (Pip) / DMF (3 x 30 min). Subsequent Fmoc deprotection was performed using 20% ​​Pip / DMF for 3 x 30 min, or 4 x 30 min for more difficult couplings. After deprotection, the resin was washed with 10 volumes of DMF for 5 x 2 min. Amino acids were preactivated using diisopropylcarbodiimide (DIC) / ethyl cyanohydroxyiminoacetate (Oxyma) in DMF at room temperature for 30 min. Coupling of the activated amino acids to the resin-bound peptide was performed for a specific time for each amino acid. After each coupling, the resin was washed with 10 volumes of DMF solvent for 5 x 2 min. To isolate the final product, the resin-bound product was washed with 10 volumes of DCM for 5 x 2 min to remove the DMF. The resin was washed with 10 volumes of IPA for 2 x 2 min to remove DCM, and then with 10 volumes of methyl tert-butyl ether (MTBE) for 5 x 2 min, and then the product was dried under vacuum at 40 ° C. The resin-bound product was refrigerated (-20 ° C). In order to analyze, the peptide was cleaved from the resin with an acidic cocktail consisting of trifluoroacetic acid (TFA) / H2O / TIPS (triisopropylsilane) / DTT (dithiothreitol) in the following ratios (0.93v / 0.04v / 0.03v / 0.03w). The resin was swollen with DCM (4-5mL, 3x30min) and drained. The cleavage mixture (4-5mL) was added to the pre-swollen resin, and the suspension was stirred at room temperature for 2 hours. The solution was filtered, then the resin was washed with a small amount of DCM and combined with the cleavage solution. The resulting solution was poured into 7-10 volumes of cold (0 ° C) methyl tert-butyl ether (MTBE). The suspension was aged at 0°C for 30 minutes, the resulting precipitate was centrifuged, and the clear solution was decanted. The residue was suspended in an equal volume of MTBE, centrifuged again, and the resulting suspension was decanted. After decanting, the clear MTBE solution containing the precipitated peptide was dried overnight at 40°C under vacuum.

[0105] Compound 1 was synthesized by Method 1

[0106]

[0107] Compound 1 can be prepared essentially by the method described in WO2020 / 159949.

[0108] Eicosanedioic acid mono(1,1-dimethylethyl) ester (15.0 kg, limiting reagent) and N-hydroxysuccinimide (1.2 equiv) were dissolved in ethyl acetate at 27°C. A solution of DCC (1.25 equiv) in ethyl acetate was added, and the reaction was stirred at 22°C for 24 hours. The resulting DCU byproduct was filtered off, and the organic phase was then extracted three times with 5% aqueous NaCl solution. After extraction, the organic phase was concentrated, co-evaporated with isopropanol, and then heptane was added for crystallization. After filtration, the filter cake was rinsed with heptane and dried at 25°C to obtain 17.0 kg of INT1 with a yield of 87% and a purity of 99%.

[0109] At 40 ° C, H-Glu-OtBu (7.7 kg, 1.1 equivalents) was dissolved in DCM (54 L), and then a solution of TMSA (11.3 kg) dissolved in DCM (7 L) was added, and the reaction mixture was stirred at 40 ° C for 1 hour. INT1 (17.0 kg) DCM solution was added at room temperature and stirred for 8 hours. After the reaction was completed, DCM was exchanged for ethyl acetate by distillation. The organic phase was washed three times with a 2% KHSO4 / NaCl aqueous solution and then washed four times with a 2% NaCl aqueous solution. After separating and removing the aqueous phase, the organic phase was concentrated with isopropanol, diluted with isopropanol, and then water was added for crystallization. After filtration, the filter cake was washed with a water / isopropanol mixture and then dried at 30 ° C to obtain 17.3 kg INT2 with a yield of 86% and a purity of 99%.

[0110] At 27 ° C, INT2 (17.3 kg) and N-hydroxysuccinimide (4.1 kg, 1.2 equivalents) were dissolved in ethyl acetate (336 kg). A solution of DCC (8.33 kg, 1.25 equivalents) in ethyl acetate was added, and the reaction was stirred at 22 ° C for 24 hours. The resulting DCU by-product was filtered off. The organic phase was concentrated, co-evaporated with isopropanol, and then crystallized by cooling the isopropanol solution (~125 L). Afterwards, the filter cake was rinsed with cold isopropanol and dried at 25 ° C to obtain 16.3 kg of INT3 with a yield of 81% and a purity of 96%.

[0111] At 22 ° C, 17-amino-10-oxo-3,6,12,15-tetraoxa-9-azaheptadecanoic acid (AEEA2) (8.1 kg, 26.3 mol) was suspended in DCM (54 L), a solution of TMSA (7.68 kg, 59.9 mol) in DCM (6.2 L) was added, and the reaction mixture was stirred at 40 ° C for 1 hour. INT3 (16 kg) was suspended in DCM (31 L) at 35 ° C and then added to the TMS-protected (AEEA2) mixture at 22 ° C. The reactants were stirred for 12 hours, and after the reaction was complete, the mixture was concentrated and then exchanged into ethyl acetate. The organic phase was washed three times with 2% KHSO4 / NaCl aqueous solution (~200 L), and then washed 4 times with 2% NaCl aqueous solution (~200 L) until the target pH value was 4.5. The organic phase was concentrated and exchanged into acetonitrile. The acetonitrile solution was cooled to -20 ° C, and the resulting suspension was aged at -20 ° C for 15 hours. The mixture was filtered and the filter cake was rinsed with cold acetonitrile and then dried at <0°C to give 18.4 kg of Preparation 6 (88% yield) with a purity of 96%. The overall yield was 53%.

[0112] Compound 1 was synthesized by method 2

[0113] Alternatively, Formulation 6 can be prepared by solid phase peptide synthesis using a peptide synthesizer.

[0114] Standard coupling procedures were employed.

[0115] Standard coupling conditions:

[0116] 0.133 M, 2.0 eq. HATU, 5.0 eq. DIEA, ambient temperature, 3 hours, deprotected with 20% piperidine / DMF 3 x 15 min.

[0117] Resin addition:

[0118] FmocNH-AEEA on 2-CTC resin (0.99 mmol / g): 1.01 g in each parallel reaction.

[0119] The automated program used DMF swelling, followed by Pip / DMF; DMF wash; mixing with amino acids, DIEA, HATU; and DMF wash cycles, followed by drying.

[0120] By stirring the combined lots 1.5 hours in 30% HFIP / DCM (240mL), cracking resin.Filter, wash resin, and remove solvent from filtrate in a vacuum. The gained oily matter is dissolved in acetonitrile and removed solvent again. This operation obtains 30.47g (146% of theoretical yield) of viscous yellow oily matter, which contains 52.3 area % of the required product by UPLC analysis. The crude product is purified by flash chromatography (500 grams of silica gel, eluted with 85% DCM / 10% methanol / 5% acetic acid, collecting 38x100mL fractions). The concentrate (17.94g) of previous chromatographic separation is crystallized to obtain 13.4g (productive rate 74.7%) product, and UPLC purity is 91.65 area %.

[0121] Example 1

[0122] Example (Linear SPPS)

[0123] SEQ ID NO: 1 can be prepared essentially by the method described in WO2020 / 159949, except that lysine is added at position 20 in SEQ ID NO: 1 using Fmoc-L-Lys(mtt)-OH instead of Fmoc-L-Lys(IVDde)-OH:

[0124]

[0125] SEQ ID NO: 1 (Telporin linear 1-39 with Lys-20mtt protection on resin) (2.0 g) 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 treated again with 15 ml of 30% HFIP / DCM, mixed for 1 hour, and drained. The resin was washed five times with 15 ml of DCM and then dried under vacuum at 35°C to constant weight to yield SEQ ID NO: 2.

[0126]

[0127] SEQ ID NO: 2 (Telpol peptide linear 1-39 with Lys-20NH2 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 washings 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. The solution was added portionwise to 20 ml of cold water to precipitate the cleaved peptide. After cooling at 5°C for about 30 minutes, the solid was filtered, washed with 20 ml of water, and dried under vacuum at 45°C to constant weight (1.25 g) to give SEQ ID NO: 3.

[0128]

[0129] To a solution of SEQ ID NO: 3 (Telpotide 1-39Lys-20NH2 linear peptide) (500 mg, 0.091 mmol), compound 1 (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 hours. HPLC analysis showed complete conversion of the linear peptide to the desired product. The solution was added portionwise 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 through a sintered filter, washed with 20 ml of water, and dried under vacuum to yield 535 mg of protected telpotide (SEQ ID NO: 4).

[0130]

[0131] The protected tilpoitide (SEQ ID NO: 4) can be substantially deprotected by the method described in WO2020 / 159949 to obtain tilpoitide (SEQ ID NO: 5):

[0132]

[0133] Alternatively, after removing mtt from SEQ ID NO: 1 to form SEQ ID NO: 2, Compound 1 is coupled to SEQ ID NO: 2 to form SEQ ID NO: 6.

[0134]

[0135] The resin is then removed from SEQ ID NO: 6 to form SEQ ID NO: 4, which is globally deprotected to form telpol peptide (SEQ ID NO: 5).

[0136] Example 2 MTT method-2

[0137] Removal of the mtt group from Lys-20 of the TZP1-39 linear peptide:

[0138] Lys-20mtt-protected TZP linear 1-39 (2.0 g) on ​​resin was suspended in 15 ml of 30% (v / v) HFIP / DCM in a 45 ml SymphonyX reactor. The reaction was mixed at ambient temperature for 1 hour and then drained. The resin was treated again with 15 ml of 30% HFIP / DCM, mixed for 1 hour, and drained. The resin was washed five times with 15 ml of DCM each time and then dried under vacuum at 35°C to constant weight.

[0139]

[0140] Soft cleavage of TZP 1-39Lys-20 NH2 linear peptide from Sieber resin:

[0141] TZP linear I-39 (2.0 g) with Lys-20NH2 on Sieber resin 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 sintered filter and rinsed with 10 ml of DCM. The combined filtrate and washings 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. The solution was added to 20 ml of cold water in several portions, resulting in precipitation of the cleaved peptide. After cooling at 5°C for about 30 minutes, the solid was filtered, washed with 20 ml of water, and dried under vacuum at 45°C to constant weight (1.25 g).

[0142]

[0143]

[0144] The TZP1-39 Lys-20 NH2 linear peptide was coupled to (S)-22-(tert-butyloxycarbonyl)-45,45-dimethyl-10,19,24,43-tetraoxo-3,6,12,15,44-pentaoxa-9,18,23-triazatetrahexadecanoic acid:

[0145] To a solution of TZP 1-39Lys-20 NH2 linear peptide (500 mg, 0.091 mmol), (S)-22-(tert-butyloxycarbonyl)-45,45-dimethyl-10,19,24,43-tetraoxo-3,6,12,15,44-pentaoxa-9,18,23-triazatetrahexadecanoic acid (2.0 equiv.), and 141.3 mg (2.0 equiv.) of PyBOP in 5 ml of DMF was added DIEA (48 ml, 3.0 equiv.). The reaction solution was stirred at ambient temperature for 18 hours. HPLC analysis indicated complete conversion of the linear peptide to the desired product. The solution was added portionwise 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 through a sintered filter, washed with 20 ml of water, and dried under vacuum to yield 535 mg of protected tirpol peptide (SEQ ID NO: 4).

[0146] The protected tirpotide (SEQ ID NO: 4) can be substantially deprotected by the method described in WO2020 / 159949 to obtain tirpotide (SEQ ID NO: 5)

[0147] Determination of chiral purity

[0148] The structures of the tirpotide fragments, including the amino acid sequences (residue numbers represent their positions in the fully assembled tirpotide drug substance), were prepared essentially as described herein. The L- and D-isomer fragment materials were synthesized by Eli Lilly (Indianapolis, IN USA) as high-purity amorphous solids. The fragment samples were fully characterized for identity, purity, and content. The tirpotide fragments are "protected" peptides, in which the active side chains of the amino acids (e.g., hydroxyl, carboxyl, amino, etc.) are protected by functional groups (e.g., Fmoc, trityl (Trt), tert-butyloxycarbonyl (Boc), and tert-butyl (tBu)). The L-isomer fragment material was also analyzed for chiral purity using established GCMS and LCMS methods (Kalikova et al., Separations 3, pp. 30-48 (2016); Liardon et al., J. Chromatogr. A 203, pp. 385-395 (1981)).

[0149] sequence

[0150] SEQ ID NO:5

[0151] Tipoleptide

[0152] YX1EGTFTSDYSIX2LDKIAQKAFVQWLIAGGPSSGAPPPS

[0153] wherein X1 is Aib; X2 is Aib; K is replaced at position 20 by (2-[2-(2-aminoethoxy)-ethoxy]-acetyl)2-(γGlu)1-CO-(CH2) 18 -CO2H) was conjugated to the ε-amino group of the K side chain for chemical modification; the C-terminal amino acid was amidated to form a C-terminal primary amide.

Claims

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

2. The compound as claimed in claim 1, wherein the compound is:

3. A method for preparing telpotide (SEQ ID NO: 5), comprising a. deprotecting the compound of SEQ ID NO: 1; b. Remove the resin; c. coupling SEQ ID NO: 3 with compound 1; and d. Deprotecting SEQ ID NO:

4.

4. A method as claimed in claim 3, wherein Deprotecting the compound of SEQ ID NO: 1 to obtain the compound of (SEQ ID NO: 2); removing the resin to obtain (SEQ ID NO: 3); SEQ ID NO: 3 was coupled with compound 1 to give (SEQ ID NO: 4).

5. A method for preparing tilpoxetine (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); and d. Deprotecting SEQ ID NO: 4 to obtain telpotide (SEQ ID NO: 5).

6. A continuous flow method for preparing tilpoxetine (SEQ ID NO: 5), comprising: a. deprotecting the compound of SEQ ID NO: 1; b. coupling SEQ ID NO: 2 with compound 1; c. Deprotection and resin removal; nanofiltration is used online to purify and provide solvent exchange.

7. A continuous flow method as in c, wherein chromatography is used on-line.

8. A continuous flow method for preparing telpol peptide (SEQ ID NO: 5), comprising a. deprotecting the compound of SEQ ID NO: 1; b. removing the resin and optionally placing chromatography and nanofiltration online; c. coupling SEQ ID NO: 3 with compound 1; placing nanofiltration online; d. Online chromatography and nanofiltration were performed to deprotect SEQ ID NO: 4.

Images