Trophynetide composition

Abstract

To provide a composition of trophinetide. [Solution] This disclosure describes a compound of formula (I), its stereoisomers, by-compounds, pharmaceutical compositions, and methods for producing such compounds using silylating agents, as well as methods for producing compositions and products produced using such methods. More specifically, this disclosure describes the production of trophinetides and by-products, compositions, and products containing such compounds for pharmaceutical applications for the treatment of neurodegenerative or neurodevelopmental disorders. JPEG2026063085000110.jpg55147

Description

[Technical Field]

[0001] [Technical field] This disclosure provides compositions comprising trofinetide (glycyl-L-2-methylprolyl-L-glutamic acid, or "G-2-MePE") and methods for producing them. The compositions are produced using a novel method and include trofinetide and products of other synthetic methods. [Background technology]

[0002] [Background technology] Trophinetide is a synthetic compound with a core structure similar to glycyl-prolyl-glutamic acid (or "GPE"). Trophinetide has been found to be useful in the treatment of neurodegenerative conditions, and more recently, it has been found to be effective in the treatment of autism spectrum disorder and neurodevelopmental disorders. [Overview of the Initiative] [Means for solving the problem]

[0003] [Overview of the prefecture] The inventors have identified a new problem in this field, namely the difficulty in scaling up current methods for producing compositions containing trophinetide to a commercial scale.

[0004] To address this problem, the inventors have identified novel compositions and methods of production in which a compound, or a salt or stereoisomer or hydrate thereof, is produced using a silylation technique. Using this novel method, novel compositions and substantially large quantities of trophinetide are produced, resulting in commercially useful compositions, kits, and products containing trophinetide.

[0005] This disclosure relates to compounds of formula (I). [ka] or its stereoisomers, hydrates, or pharmaceutically acceptable salts, and compounds of formula (II). [Chemical formula] or a stereoisomer, hydrate, or pharmaceutically acceptable salt thereof, and / or a compound of formula (III) [Chemical formula] comprising a composition comprising or a stereoisomer, hydrate, or pharmaceutically acceptable salt thereof wherein R1, R2, R3 and R4 are independently selected from the group consisting of hydrogen and C 1-4 alkyl, provided that at least one of R1, R2, R3 and R4 is C 1-4 alkyl.

[0006] The present disclosure also relates to a compound according to formula (Ia) (trophinetide) [Chemical formula] or a hydrate or pharmaceutically acceptable salt thereof.

[0007] The present disclosure also relates to a composition comprising trophinetide, said composition being a) coupling H-MePro-OH and Z-Gly-OH in the presence of an activator, a silylating agent and a solvent, or b) coupling Z-Gly-OH and Suc-OH in the absence or presence of an activator and in the absence or presence of a silylating agent, and then coupling the resulting Z-Gly-OSu and H-MePro-OH, or c) coupling the resulting Z-Gly-MePro-OH and H-Glu-OH in the presence of an activator, a silylating agent and a solvent, or d) obtaining Z-Gly-MePro-Glu-OH, and e) deprotecting Z-Gly-MePro-Glu-OH to obtain a composition comprising trophinetide.

[0008] The present disclosure also provides the following steps a) coupling H-MePro-OH and Z-Gly-OH in the presence of an activator, a silylating agent and a solvent, or b) coupling Z-Gly-OH and Suc-OH in the absence or presence of a solvent and in the absence or presence of a silylating agent, and then coupling the resulting Z-Gly-OSu and H-MePro-OH c) coupling the resulting Z-Gly-MePro-OH and H-Glu-OH in the presence of an activator, a silylating agent and a solvent d) obtaining Z-Gly-MePro-Glu-OH e) deprotecting Z-Gly-MePro-Glu-OH to obtain a composition containing trofinetide, and also includes a method for producing a composition containing trofinetide.

[0009] The present disclosure also provides a dosage form comprising a compound of trofinetide and a compound of formula (IIa), [Chemical formula] or a hydrate or pharmaceutically acceptable salt thereof, and also includes a kit including instructions for use.

[0010] The present disclosure also provides a dosage form comprising a compound of formula (Ia) and a compound of formula (IIa), and also includes a kit comprising one or more compounds selected from the group consisting of formula (III), formula (IIIa), formula (IV), formula (V), formula (VI), formula (VII), formula (VIII) and formula (IX).

Embodiments for Carrying out the Invention

[0011] Peptide synthesis using persilylation generally involves the selection of a peptide or amino acid, followed by several steps. In the first step, the peptide or amino acid is reacted with a persilylation agent, optionally in an organic solvent. It may be desirable to use a persilylation agent selected from the group consisting of N,O-bis(trimethylsilyl)acetamide (BSA), N,O-bis(trimethylsilyl)trifluoroacetamide, hexamethyldisilazane, N-methyl-N-(trimethylsilyl)acetamide (TMA), N-methyl-N-(trimethylsilyl)trifluoroacetamide, N-(trimethylsilyl)acetamide, N-(trimethylsilyl)diethylamine, N-(trimethylsilyl)dimethylamine, 1-(trimethylsilyl)imidazole, and 3-(trimethylsilyl)2-oxazolidone.

[0012] usefulness Trophinetide has been shown to be useful in the treatment of neurodegenerative conditions, neurodevelopmental disorders, and autism spectrum disorder.

[0013] definition The following definitions should be understood as they are given unless specifically defined elsewhere in this disclosure.

[0014] The terms "Cbz" and "Z" refer to benzyloxycarbonyl protecting groups, are interchangeable, and have the following structures: [ka]

[0015] The term "trophinetide" refers to glycyl-L-2-methylprolyl-L-glutamic acid of formula (Ia), or "G-2-MePE", or "H-Gly-MePro-Glu-OH", or "Gly-MePro-Glu-OH", and the chiral center has an S,S configuration. [ka]

[0016] The IUPAC name for trophinetide is (2S)-2-[[(2S)-1-(2-aminoacetyl)-2-methylpyrrolidine-2-carbonyl]amino]pentanedioic acid.

[0017] The term "approximately" means the mean of the variable ± 20% of the mean of the variable.

[0018] The term "eq" means "equivalent." Unless otherwise specified, all equivalents (eq) are expressed in molar terms with respect to the limiting reagent.

[0019] The terms "vol" and "vols" mean "capacity" and "capacity," respectively.

[0020] The term "DIPEA" refers to N,N-diisopropylethylamine, or Hünig base.

[0021] The term "DMAC" refers to N,N-dimethylacetamide.

[0022] The term "NMT" means "below" or "less than".

[0023] The term "TMA" stands for N-methyl-N-(trimethylsilyl)acetamide.

[0024] The term "BSA" stands for N,O-bis(trimethylsilyl)acetamide.

[0025] The term "Oxymapure" refers to ethyl 2-cyano-2-(hydroxyimino)acetate, or ethyl (hydroxyimino)cyanoacetate.

[0026] The term "IPE" refers to isopropyl ether or diisopropyl ether.

[0027] The term "ACN" refers to acetonitrile.

[0028] The term "NLT" means "Near" or "Near".

[0029] The term "MTBE" refers to 2-methoxy-2-methylpropane, or methyl-tert-butyl ether.

[0030] The term "iPrOH" means propane-2-ol, or isopropyl alcohol, or isopropanol.

[0031] The term "iPrOAc" refers to isopropyl acetate.

[0032] The term "UPLC" stands for ultra-high-performance liquid chromatography.

[0033] The term "Piv-Cl" refers to pivaloyl chloride.

[0034] The terms "¼" or "AcOEt" refer to ethyl acetate.

[0035] The term "HPLC" stands for High Performance Liquid Chromatography.

[0036] The term "MeOH" refers to methyl alcohol or methanol.

[0037] The term "Pd / C" refers to a palladium-carbon catalyst.

[0038] The term "Pd / Si" refers to a palladium silica catalyst.

[0039] The term "RT" refers to ambient temperature, which is typically 25°C.

[0040] The term "LC" stands for liquid chromatography.

[0041] The term "RRT" refers to relative retention time.

[0042] The term "TFA" refers to trifluoroacetic acid.

[0043] The term "H-MePro-OH" refers to (2S)-2-methylpyrrolidine-2-carboxylic acid in the form of a base or hydrochloride salt.

[0044] The term "MePro.HCl" refers to (2S)-2-methylpyrrolidine-2-carboxylate hydrochloride.

[0045] The term "Z-Gly-OH" refers to benzyloxycarbonylglycine, or Cbz-glycine.

[0046] The term "H-Glu-OH" refers to (2S)-2-aminopentanediic acid, or L-glutamic acid.

[0047] The term "EDC.HCl" means 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, or N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride.

[0048] The term "Z-Gly-MePro-OH" means ((S)-1-(((benzyloxy)carbonyl)glycyl)-2-methylpyrrolidine-2-carboxylic acid), or N-benzyloxycarbonyl-glycyl-L-2-methylproline.

[0049] The term "Z-Gly-MePro-Glu-OH" means ((S)-1-(((benzyloxy)carbonyl)glycyl)-2-methylpyrrolidine-2-carbonyl)-L-glutamic acid, or N-benzyloxycarbonyl-glycyl-L-2-methylprolyl-L-glutamic acid.

[0050] The term "Et-Gly-MePro-Glu-OH" means ((S)-1-(ethylglycyl)-2-methylpyrrolidine 2-carbonyl)-L-glutamic acid, or N-ethylglycyl-L-2-methylprolyl-L-glutamic acid.

[0051] The term "Me-Gly-MePro-Glu-OH" means ((S)-1-(methylglycyl)-2-methylpyrrolidine-2-carbonyl)-L-glutamic acid, or methyl-glycyl-L-2-methylprolyl-L-glutamic acid.

[0052] The term "Me2-Gly-MePro-Glu-OH" means ((S)-1-(dimethylglycyl)-2-methylpyrrolidine-2-carbonyl)-L-glutamic acid, or N,N-dimethylglycyl-L-2-methylprolyl-L-glutamic acid.

[0053] The term "Gly-MePro-Glu(OiPr)-OH" means glycyl-L-2-methylprolyl-L-glutamic acid 5-isopropyl ester, or glycyl-L-2-methylprolyl-L-glutamic acid γ-isopropyl ester.

[0054] The term "Gly-MePro-Glu-OiPr" means glycyl-L-2-methylprolyl-L-glutamic acid 1-isopropyl ester, or glycyl-L-2-methylprolyl-L-glutamic acid α-isopropyl ester.

[0055] The term "Gly-MePro-Glu(OiPr)-OiPr" refers to glycyl-L-2-methylprolyl-glutamic acid 1,5-diisopropyl ester.

[0056] The term "Gly-MePro-Glu(OMe)-OH" means glycyl-L-2-methylprolyl-L-glutamic acid 5-methyl ester, or glycyl-L-2-methylprolyl-L-glutamic acid γ-methyl ester.

[0057] The term "Gly-MePro-Glu-OMe" means glycyl-L-2-methylprolyl-L-glutamic acid 1-methyl ester, or glycyl-L-2-methylprolyl-L-glutamic acid α-methyl ester.

[0058] The term "Gly-MePro-Glu(OMe)-OMe" refers to glycyl-L-2-methylprolyl-L-glutamic acid 1,5-dimethyl ester.

[0059] The term "Gly-MePro-Glu(OEt)-OH" means glycyl-L-2-methylprolyl-L-glutamate 5-ethyl ester, or glycyl-L-2-methylprolyl-L-glutamate γ-ethyl ester.

[0060] The term "Gly-MePro-Glu-OEt" means glycyl-L-2-methylprolyl-L-glutamate 1-ethyl ester, or glycyl-L-2-methylprolyl-L-glutamate α-ethyl ester.

[0061] The term "Gly-MePro-Glu(OEt)-OEt" refers to glycyl-L-2-methylprolyl-L-glutamic acid 1,5-diethyl ester.

[0062] The term "Suc-OH" refers to N-hydroxysuccinimide. The term "Z-Gly-OSu" refers to benzyloxycarbonyl-glycine N-succinimidyl ester.

[0063] The term "DCC" refers to N,N'-dicyclohexylcarbodiimide.

[0064] The term "DCU" refers to 1,3-dicyclohexylurea.

[0065] The term "TEA" refers to triethylamine.

[0066] The term "CH2Cl2" refers to DCM, or dichloromethane.

[0067] The term "NaHCO3" means bicarbonate.

[0068] The term "iButOH" means butan-2-ol, or isobutyl alcohol, or isobutanol.

[0069] The term "DMAPA" refers to dimethylaminopropylamine.

[0070] The term "KHSO4" refers to potassium bisulfate.

[0071] The term "H2O" means water.

[0072] The term "NaCl" means sodium chloride or salt.

[0073] The term "DMA" stands for N,N-dimethylacetamide.

[0074] The term "DMF" stands for N,N-dimethylformamide.

[0075] The term "HCl" refers to hydrochloric acid.

[0076] The term "HOBt" refers to 1-hydroxybenzotriazole.

[0077] The term "HOAt" refers to 1-hydroxyazabenzotriazole.

[0078] The term "TBTU" refers to 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminiumtetrafluoroborate.

[0079] The term "NMM" stands for N-methylmorpholine.

[0080] The term "ICH" refers to the International Conference on Harmonisation of Regulations for Pharmaceuticals for Human Use.

[0081] The term "NMR" stands for Nuclear Magnetic Resonance.

[0082] The term "UV" refers to ultraviolet light.

[0083] The term "MS" stands for mass spectrometry.

[0084] The term "gm" stands for grams.

[0085] The term "μl" means microliter.

[0086] The terms "activator" and "carboxyl group activator" refer to reagents that promote the coupling of two amino acids and / or peptides by the formation of an amide bond. Examples of activators and carboxyl group activators include carbodiimides, acyl halides, phosphonium salts and uronium or guanidinium salts, acyl halides such as isobutyl chloroformate or pivaloyl chloride, or carbodiimides such as EDC.HCl or DCC.

[0087] The term "comprising" means "including, but not limited to."

[0088] The term "consisting of" means "containing only the listed materials."

[0089] The phrase "consisting essentially of" means "including the listed materials and their equivalents."

[0090] The terms "%w / w" and "wt%" are used interchangeably throughout this disclosure.

[0091] "Specified amount" means the amount of compound, expressed in units of mass / volume or mass / mass, that is intended to be present in the product. The actual amount of compound may exceed 100 wt% of the specified amount, for example, if an excess is included to compensate for losses of the compound during storage due to decomposition or degradation of the compound.

[0092] The terms "persilylation agent" or "silylation agent" refer to compounds that add a silicon atom to a peptide or amino acid.

[0093] The term "supersilylation" refers to a compound in which a group having an active hydrogen atom capable of reacting with a silylating agent is sufficiently silylated to obtain a homogeneous reaction medium.

[0094] The term "silylated functional group" is understood to specifically refer to a group that has an active hydrogen atom that reacts with a silylating agent, such as an amino group, hydroxyl group, mercapto group, or carboxyl group.

[0095] The term "by-product(s)" refers to the compounds of formulas (II), (IIa), (III), (IIIa), (IV), (V), (VI), (VII), (VIII), and (IX).

[0096] overview This disclosure includes a description of a method for producing products comprising compounds of formula (I), and compounds of formula (II), and / or formula (III), and / or formula (IV), and / or formula (V), and / or formula (VI), and / or formula (VII), and / or formula (VIII), and / or formula (IX), as well as compositions containing one or more compounds of these formulas. The general method disclosed involves the synthesis of compounds using a silylating agent. Based on the chirality of the starting materials, a mixture of compounds can be produced, the majority of which have the chirality of the starting materials, and some additional compounds having different chiralities.

[0097] These compounds and compositions are useful in the treatment of various neurodegenerative disorders, autism spectrum disorders, and neurodevelopmental disorders.

[0098] Compounds of the Disclosure The following compounds are considered independent of each other, but can be combined with any other compound. They include the compound of formula (I), and the compounds of formulas (II), (IIa), (III), (IIIa), (IV), (V), (VI), (VII), (VIII), and (IX).

[0099] Compound of formula (I) [ka] or its hydrate or pharmaceutically acceptable salt, wherein the chiral center has one of the following configurations: S,S, S,R, R,S, or R,R, and the chiral center is denoted by * in formula (I). The chiral centers of formulas (Ia), (II), (IIa), (III), (IIIa), (IV), (V), (VI), (VII), (VIII), and (IX) are the same as those of formula (I).

[0100] Compound of formula (Ia) [ka] or a hydrate or pharmaceutically acceptable salt thereof, wherein the chiral center has an S,S configuration.

[0101] Compound of formula II [ka] or a hydrate or pharmaceutically acceptable salt thereof, wherein the chiral center has one of the following configurations: S,S, S,R, R,S, or R,R.

[0102] Compound of formula (IIa) [ka] or a hydrate or pharmaceutically acceptable salt thereof, wherein the chiral center has an S,S configuration.

[0103] Compound of formula (III) [ka] or a hydrate or pharmaceutically acceptable salt thereof, wherein the chiral center has one of the following configurations: S,S, S,R, R,S, or R,R, and R1, R2, R3, and R4 are independently hydrogen and C 1-4 Selected from the group consisting of alkyl groups, wherein at least one of R1, R2, R3, and R4 is C 1-4 It is alkyl.

[0104] A compound of formula (III) containing Gly-MePro-Glu(OMe)-OH, which means glycyl-L-2-methylprolyl-L-glutamic acid 5-methyl ester or glycyl-L-2-methylprolyl-L-glutamic acid γ-methyl ester.

[0105] A compound of formula (III) comprising Gly-MePro-Glu-OMe, which means glycyl-L-2-methylprolyl-L-glutamic acid 1-methyl ester or glycyl-L-2-methylprolyl-L-glutamic acid α-methyl ester.

[0106] The compound of formula (III) contains Gly-MePro-Glu(OMe)-OMe, which means glycyl-L-2-methylprolyl-L-glutamic acid 1,5-dimethyl ester.

[0107] A compound of formula (III) comprising Gly-MePro-Glu(OEt)-OH, which means glycyl-L-2-methylprolyl-L-glutamate 5-ethyl ester or glycyl-L-2-methylprolyl-L-glutamate γ-ethyl ester.

[0108] A compound of formula (III) comprising Gly-MePro-Glu-OEt, which means glycyl-L-2-methylprolyl-L-glutamate 1-ethyl ester or glycyl-L-2-methylprolyl-L-glutamate α-ethyl ester.

[0109] The compound of formula (III) includes Gly-MePro-Glu(OEt)-OEt, which means glycyl-L-2-methylprolyl-L-glutamic acid 1,5-diethyl ester.

[0110] Compound of formula (IIIa) [ka] or a hydrate or pharmaceutically acceptable salt thereof, wherein the chiral center has an S,S configuration.

[0111] Compound of formula (IV) [ka] or a hydrate or pharmaceutically acceptable salt thereof, wherein the chiral center has one of the following configurations: S,S, S,R, R,S, or R,R.

[0112] Compound of formula (V) [ka] or a hydrate or pharmaceutically acceptable salt thereof, wherein the chiral center has one of the following configurations: S,S, S,R, R,S, or R,R.

[0113] Compound of formula (VI) [ka] or a hydrate or pharmaceutically acceptable salt thereof, wherein the chiral center has one of the following configurations: S,S, S,R, R,S, or R,R.

[0114] Compound of formula (VII) [ka] or a hydrate or pharmaceutically acceptable salt thereof, wherein the chiral center has one of the following configurations: S,S, S,R, R,S, or R,R.

[0115] Compound of formula (VIII) [ka] or a hydrate or pharmaceutically acceptable salt thereof, wherein the chiral center has one of the following configurations: S,S, S,R, R,S, or R,R.

[0116] Compound of formula (IX) [ka] or a hydrate or pharmaceutically acceptable salt thereof, wherein the chiral center has one of the following configurations: S,S, S,R, R,S, or R,R.

[0117] The compounds of formulas (II), (IIa), (III), (IIIa), (IV), (V), (VI), (VII), (VIII), and (IX) are, if present, by-products of the compositions disclosed herein.

[0118] Compositions, manufactures, and products of this disclosure The products of this disclosure refer to non-isolated materials for use, isolated materials, chemical intermediates, formulations, dosage forms, kits, instructions, etc., including the compositions of this disclosure. The compositions and products of this disclosure include the following:

[0119] A composition comprising a compound of formula (I), its stereoisomer, hydrate, or pharmaceutically acceptable salt, and a compound of formula (II), its stereoisomer, hydrate, or pharmaceutically acceptable salt.

[0120] A composition comprising the compound of formula (I), its stereoisomer, hydrate, or pharmaceutically acceptable salt; the compound of formula (II), its stereoisomer, hydrate, or pharmaceutically acceptable salt; and / or the compound of formula (III), its stereoisomer, hydrate, or pharmaceutically acceptable salt.

[0121] A composition comprising a compound of formula (I), its stereoisomer, hydrate, or pharmaceutically acceptable salt; a compound of formula (II), its stereoisomer, hydrate, or pharmaceutically acceptable salt; and / or a compound of formula (IX), its stereoisomer, hydrate, or pharmaceutically acceptable salt.

[0122] A composition comprising a compound of formula (Ia) (trophinetide), its hydrate, or a pharmaceutically acceptable salt thereof, and at least a compound of formula (IIa), its hydrate, or a pharmaceutically acceptable salt thereof.

[0123] A composition comprising the compound of formula (Ia) (trophinetide) and the compound of formula (IIa).

[0124] A composition comprising the compound of formula (Ia) (trophinetide), its hydrate, or a pharmaceutically acceptable salt thereof, and the compound of formula (IIa), its hydrate, or a pharmaceutically acceptable salt thereof, and / or the compound of formula (IIIa), its hydrate, or a pharmaceutically acceptable salt thereof.

[0125] A composition comprising the compound of formula (Ia) (trophinetide), its hydrate, or a pharmaceutically acceptable salt thereof, and the compound of formula (IIa), its hydrate, or a pharmaceutically acceptable salt thereof, and / or the compound of formula (III), its hydrate, or a pharmaceutically acceptable salt thereof.

[0126] A composition comprising a compound of formula (Ia) (trophinetide), a compound of formula (II), and / or a compound of formula (III).

[0127] A composition comprising the compound of formula (I), its stereoisomer, hydrate, or pharmaceutically acceptable salt; the compound of formula (II), its stereoisomer, hydrate, or pharmaceutically acceptable salt; and / or the compound of formula (IV), its stereoisomer, hydrate, or pharmaceutically acceptable salt.

[0128] A composition comprising a compound of formula (I), its stereoisomer, hydrate, or pharmaceutically acceptable salt; a compound of formula (II), its stereoisomer, hydrate, or pharmaceutically acceptable salt; and / or a compound of formula (V), its stereoisomer, hydrate, or pharmaceutically acceptable salt.

[0129] A composition comprising the compound of formula (I), its stereoisomer, hydrate, or pharmaceutically acceptable salt; the compound of formula (II), its stereoisomer, hydrate, or pharmaceutically acceptable salt; and / or the compound of formula (VI), its stereoisomer, hydrate, or pharmaceutically acceptable salt.

[0130] A composition comprising the compound of formula (I), its stereoisomer, hydrate, or pharmaceutically acceptable salt; the compound of formula (II), its stereoisomer, hydrate, or pharmaceutically acceptable salt; and / or the compound of formula (VII), its stereoisomer, hydrate, or pharmaceutically acceptable salt.

[0131] A composition comprising the compound of formula (I), its stereoisomer, hydrate, or pharmaceutically acceptable salt; the compound of formula (II), its stereoisomer, hydrate, or pharmaceutically acceptable salt; and / or the compound of formula (VIII), its stereoisomer, hydrate, or pharmaceutically acceptable salt.

[0132] A composition comprising a compound of formula (I), its stereoisomers, hydrates, or pharmaceutically acceptable salts, and a compound of formula (III).

[0133] A composition comprising a compound of formula (Ia), its hydrate, or a pharmaceutically acceptable salt thereof, and a compound of formula (IIIa).

[0134] A kit containing dosage forms of the compound of formula (Ia) (trophinetide) and the compound of formula (IIa), as well as instructions for use.

[0135] The kit may further contain one or more compounds selected from the group consisting of formulas (III), (IIIa), (IV), (V), (VI), (VII), (VIII), and (IX).

[0136] Embodiment Compound of formula (I) [ka] or its stereoisomers, hydrates, or pharmaceutically acceptable salts, and compounds of formula (II). [ka] or its stereoisomers, hydrates, or pharmaceutically acceptable salts, and / or compounds of formula (III). [ka] or its stereoisomers, hydrates, or pharmaceutically acceptable salts, wherein R1, R2, R3, and R4 are independently hydrogen and C 1-4 Selected from the group consisting of alkyl groups, wherein at least one of R1, R2, R3, and R4 is C 1-4 A composition of any of the following embodiments, wherein the composition is alkyl.

[0137] The compound according to formula (I) is the same as the compound according to formula (Ia). [ka] The composition according to any preceding or succeeding embodiment, or a hydrate or pharmaceutically acceptable salt thereof.

[0138] A composition according to any preceding or succeeding embodiment, wherein the compound of formula (II), or its stereoisomers, hydrates, or pharmaceutically acceptable salts, is absent or present in an amount between approximately 0.001 ± 0.0002 wt% and approximately 2 ± 0.4 wt%, and the compound of formula (III), or its stereoisomers, hydrates, or pharmaceutically acceptable salts, is absent or present in an amount between approximately 0.001 ± 0.0002 wt% and approximately 2 ± 0.4 wt%, provided that at least one of the compounds of formula (II) or (III), or their stereoisomers, hydrates, or pharmaceutically acceptable salts, is present.

[0139] A composition according to any preceding or succeeding embodiment, wherein the compound of formula (II), or its stereoisomer, hydrate, or pharmaceutically acceptable salt, is present in an amount between about 0.001 ± 0.0002 wt% and about 0.3 ± 0.06 wt%.

[0140] A composition according to any preceding or succeeding embodiment, wherein the compound of formula (II), or its stereoisomer, hydrate, or pharmaceutically acceptable salt, is present in an amount between about 0.001 ± 0.0002 wt% and about 0.1 ± 0.02 wt%.

[0141] The compound of formula (II), or its stereoisomer, hydrate, or pharmaceutically acceptable salt, in amounts between approximately 0.001±0.0002 wt% and approximately 2±0.4 wt%, or between approximately 0.001±0.0002 wt% and approximately 1.5±0.3 wt%, or between approximately 0.001±0.0002 wt% and approximately 1±0.2 wt%, or approximately 0.001±0.0002 wt% A composition according to any prior or subsequent embodiment, present in an amount between t% and about 0.7±0.14 wt%, or in an amount between about 0.001±0.0002 wt% and about 0.5±0.1 wt%, or in an amount between about 0.001±0.0002 wt% and about 0.3±0.06 wt%, or in an amount between about 0.001±0.0002 wt% and about 0.2±0.04 wt%.

[0142] The compound according to formula (II) is the same as the compound according to formula (IIa). [ka] The composition according to any preceding or succeeding embodiment, or a hydrate thereof, or a pharmaceutically acceptable salt thereof.

[0143] A composition according to any preceding or succeeding embodiment, wherein the compound of formula (II), its stereoisomer, hydrate, or pharmaceutically acceptable salt thereof is not present.

[0144] A composition according to any preceding or succeeding embodiment, wherein a compound of formula (III), or its stereoisomer, hydrate, or pharmaceutically acceptable salt, is present in an amount between about 0.001 ± 0.0002 wt% and about 2.0 ± 0.4 wt%.

[0145] The composition comprises a compound of formula (III), and the compound of formula (III) is a compound of formula (IX). [ka] The composition according to any preceding or succeeding embodiment, or a hydrate thereof, or a pharmaceutically acceptable salt thereof.

[0146] A compound of formula (III) is included, and the compound according to formula (III) is a compound of formula (IIIa). [ka] The composition according to any preceding or succeeding embodiment, or a hydrate thereof, or a pharmaceutically acceptable salt thereof.

[0147] A composition according to any prior or subsequent embodiment, wherein a compound of formula (IIIa), its hydrate, or a pharmaceutically acceptable salt thereof is present in an amount between about 0.001 ± 0.0002 wt% and about 2.0 ± 0.4 wt%.

[0148] The compound of formula (IIIa), its hydrate, or a pharmaceutically acceptable salt thereof, in an amount between approximately 0.001±0.0002 wt% and approximately 2.0±0.4 wt%, or in an amount between approximately 0.001±0.0002 wt% and approximately 1.5±0.3 wt%, or in an amount between approximately 0.001±0.0002 wt% and approximately 1±0.2 wt%, or approximately 0.001±0.0002 wt% A composition according to any prior or subsequent embodiment, present in an amount between % and about 0.7 ± 0.14 wt%, or in an amount between about 0.001 ± 0.0002 wt% and about 0.5 ± 0.1 wt%, or in an amount between about 0.001 ± 0.0002 wt% and about 0.3 ± 0.06 wt%, or in an amount between about 0.001 ± 0.0002 wt% and about 0.2 ± 0.04 wt%.

[0149] A composition according to any preceding or succeeding embodiment, comprising a compound of formula (Ia) in an anhydrous basis between approximately 97 wt% and approximately 100 wt%, or instead between approximately 98 wt% and approximately 100 wt%, or between approximately 99 wt% and approximately 100 wt%.

[0150] The compound according to formula (III) is the compound according to formula (IV). [ka] The composition according to any preceding or succeeding embodiment, or a stereoisomer thereof, hydrate, or pharmaceutically acceptable salt thereof.

[0151] The compound of formula (IV), or its stereoisomers, hydrates, or pharmaceutically acceptable salts, in amounts between approximately 0.001±0.0002 wt% and approximately 2±0.4 wt%, or between approximately 0.001±0.0002 wt% and approximately 1.5±0.3 wt%, or between approximately 0.001±0.0002 wt% and approximately 1±0.2 wt%, or approximately 0.001±0.0002 wt% A composition according to any prior or subsequent embodiment, present in an amount between t% and about 0.7±0.14 wt%, or in an amount between about 0.001±0.0002 wt% and about 0.5±0.1 wt%, or in an amount between about 0.001±0.0002 wt% and about 0.3±0.06 wt%, or in an amount between about 0.001±0.0002 wt% and about 0.2±0.04 wt%.

[0152] The compound according to formula (III) is the compound according to formula (V). [ka] The composition according to any preceding or succeeding embodiment, or a stereoisomer thereof, hydrate, or pharmaceutically acceptable salt thereof.

[0153] The compound of formula (V), or its stereoisomer, hydrate, or pharmaceutically acceptable salt, in amounts between approximately 0.001±0.0002 wt% and approximately 2±0.4 wt%, or between approximately 0.001±0.0002 wt% and approximately 1.5±0.3 wt%, or between approximately 0.001±0.0002 wt% and approximately 1±0.2 wt%, or approximately 0.001±0.0002 wt% A composition according to any prior or subsequent embodiment, present in an amount between % and about 0.7 ± 0.14 wt%, or in an amount between about 0.001 ± 0.0002 wt% and about 0.5 ± 0.1 wt%, or in an amount between about 0.001 ± 0.0002 wt% and about 0.3 ± 0.06 wt%, or in an amount between about 0.001 ± 0.0002 wt% and about 0.2 ± 0.04 wt%.

[0154] The compound of formula (III) is the compound of formula (VI) [ka] The composition according to any preceding or succeeding embodiment, or a stereoisomer thereof, hydrate, or pharmaceutically acceptable salt thereof.

[0155] The compound of formula (VI), or its stereoisomers, hydrates, or pharmaceutically acceptable salts, in amounts between approximately 0.001±0.0002 wt% and approximately 2±0.4 wt%, or between approximately 0.001±0.0002 wt% and approximately 1.5±0.3 wt%, or between approximately 0.001±0.0002 wt% and approximately 1±0.2 wt%, or approximately 0.001±0.0002 wt% A composition according to any prior or subsequent embodiment, present in an amount between t% and about 0.7±0.14 wt%, or in an amount between about 0.001±0.0002 wt% and about 0.5±0.1 wt%, or in an amount between about 0.001±0.0002 wt% and about 0.3±0.06 wt%, or in an amount between about 0.001±0.0002 wt% and about 0.2±0.04 wt%.

[0156] The compound of formula (III) is the compound of formula (VII) [ka] The composition according to any preceding or succeeding embodiment, or a stereoisomer thereof, hydrate, or pharmaceutically acceptable salt thereof.

[0157] The compound of formula (VII), or its stereoisomers, hydrates, or pharmaceutically acceptable salts, in amounts between approximately 0.001±0.0002 wt% and approximately 2±0.4 wt%, or between approximately 0.001±0.0002 wt% and approximately 1.5±0.3 wt%, or between approximately 0.001±0.0002 wt% and approximately 1±0.2 wt%, or approximately 0.001±0.0002 wt% A composition according to any prior or subsequent embodiment, present in an amount between t% and about 0.7±0.14 wt%, or in an amount between about 0.001±0.0002 wt% and about 0.5±0.1 wt%, or in an amount between about 0.001±0.0002 wt% and about 0.3±0.06 wt%, or in an amount between about 0.001±0.0002 wt% and about 0.2±0.04 wt%.

[0158] The compound of formula (III) is the compound of formula (VIII) [ka] The composition according to any preceding or succeeding embodiment, or a stereoisomer thereof, hydrate, or pharmaceutically acceptable salt thereof.

[0159] The compound of formula (VIII), or its stereoisomer, hydrate, or pharmaceutically acceptable salt, in amounts between approximately 0.001±0.0002 wt% and approximately 2±0.4 wt%, or between approximately 0.001±0.0002 wt% and approximately 1.5±0.3 wt%, or between approximately 0.001±0.0002 wt% and approximately 1±0.2 wt%, or approximately 0.001±0.0002 A composition according to any prior or subsequent embodiment, present in an amount between wt% and about 0.7±0.14 wt%, or in an amount between about 0.001±0.0002 wt% and about 0.5±0.1 wt%, or in an amount between about 0.001±0.0002 wt% and about 0.3±0.06 wt%, or in an amount between about 0.001±0.0002 wt% and about 0.2±0.04 wt%.

[0160] [ka] The composition is a) Coupling H-MePro-OH with Z-Gly-OH in the presence of an activator, a silylation agent, and a solvent, or b) Coupling Z-Gly-OH with Suc-OH in the absence or presence of an activator and solvent, and in the absence or presence of a silylating agent, and then coupling the resulting Z-Gly-OSu with H-MePro-OH, c) Coupling the obtained Z-Gly-MePro-OH with H-Glu-OH in the presence of an activator, a silylation agent, and a solvent, d) To obtain Z-Gly-MePro-Glu-OH, e) Deprotecting Z-Gly-MePro-Glu-OH to obtain a composition comprising the compound of formula (Ia), and the composition according to any preceding or succeeding embodiment.

[0161] A composition according to any preceding or succeeding embodiment, wherein the obtained composition is dissolved or suspended in a solvent.

[0162] The above composition is a compound of formula (II). [ka] or further comprising its stereoisomer, hydrate, or pharmaceutically acceptable salt, and optionally a compound of formula (III). [ka] or comprising its stereoisomers, hydrates, or pharmaceutically acceptable salts, where R1, R2, R3, and R4 are independently hydrogen and C 1-4 Selected from the group consisting of alkyl groups, wherein at least one of R1, R2, R3, and R4 is C 1-4 A composition according to any of the preceding or subsequent embodiments, wherein the composition is alkyl.

[0163] A composition according to any preceding or succeeding embodiment, wherein the compound of formula (II), or its stereoisomers, hydrates, or pharmaceutically acceptable salts, is absent or present in an amount between approximately 0.001 ± 0.0002 wt% and approximately 2 ± 0.4 wt%, and the compound of formula (III), or its stereoisomers, hydrates, or pharmaceutically acceptable salts, is absent or present in an amount between approximately 0.001 ± 0.0002 wt% and approximately 2 ± 0.4 wt%, provided that at least one of the compounds of formula (II) or (III), or their stereoisomers, hydrates, or pharmaceutically acceptable salts, is present.

[0164] A composition according to any preceding or succeeding embodiment, wherein the compound of formula (II), or its stereoisomer, hydrate, or pharmaceutically acceptable salt, is present in an amount between about 0.001 ± 0.0002 wt% and about 0.3 ± 0.06 wt%.

[0165] A composition according to any preceding or succeeding embodiment, wherein the compound of formula (II), or its stereoisomer, hydrate, or pharmaceutically acceptable salt, is present in an amount between about 0.001 ± 0.0002 wt% and about 0.1 ± 0.02 wt%.

[0166] The compound of formula (II), or its stereoisomer, hydrate, or pharmaceutically acceptable salt, in amounts between approximately 0.001±0.0002 wt% and approximately 2±0.4 wt%, or between approximately 0.001±0.0002 wt% and approximately 1.5±0.3 wt%, or between approximately 0.001±0.0002 wt% and approximately 1±0.2 wt%, or approximately 0.001±0.0002 wt% A composition according to any prior or subsequent embodiment, present in an amount between t% and about 0.7±0.14 wt%, or in an amount between about 0.001±0.0002 wt% and about 0.5±0.1 wt%, or in an amount between about 0.001±0.0002 wt% and about 0.3±0.06 wt%, or in an amount between about 0.001±0.0002 wt% and about 0.2±0.04 wt%.

[0167] The compound according to formula (II) is the same as the compound according to formula (IIa). [ka] The composition according to any preceding or succeeding embodiment, or a hydrate thereof, or a pharmaceutically acceptable salt thereof.

[0168] A composition according to any preceding or succeeding embodiment, wherein the compound of formula (II), its stereoisomer, hydrate, or pharmaceutically acceptable salt thereof is not present.

[0169] A composition according to any preceding or succeeding embodiment, wherein the compound of formula (III), or its stereoisomer, hydrate, or pharmaceutically acceptable salt, is present in an amount between about 0.001 ± 0.0002 wt% and about 2.0 ± 0.4 wt%.

[0170] The composition according to any preceding or succeeding embodiment, wherein the silylation agent does not contain a cyano group.

[0171] The composition according to any preceding or succeeding embodiment, wherein the silylation agent is selected from the group consisting of N,O-bis(trimethylsilyl)acetamide, N,O-bis(trimethylsilyl)trifluoroacetamide, hexamethyldisilazane, N-methyl-N-(trimethylsilyl)acetamide, N-methyl-N-(trimethylsilyl)trifluoroacetamide, N-(trimethylsilyl)acetamide, N-(trimethylsilyl)diethylamine, N-(trimethylsilyl)dimethylamine, 1-(trimethylsilyl)imidazole, and 3-(trimethylsilyl)2-oxazolidone.

[0172] The composition according to any preceding or succeeding embodiment, wherein the solvent in step a) is a polar organic solvent or a polar aprotic organic solvent.

[0173] The composition according to any preceding or succeeding embodiment, wherein the solvent in step b) is a polar organic solvent or a polar aprotic organic solvent.

[0174] The composition according to any preceding or succeeding embodiment, wherein the solvent in step c) is a polar organic solvent or a polar aprotic organic solvent.

[0175] The composition according to any preceding or succeeding embodiment, wherein the solvent is selected from the group consisting of alkyl acetates, chlorinated hydrocarbons, alkyl cyanides, and amide solvents, or mixtures thereof.

[0176] The composition according to any preceding or succeeding embodiment, wherein the activator is selected from the group consisting of carbodiimide, acyl halide, phosphonium salt, uronium salt, and guanidinium salt.

[0177] The composition according to any preceding or subsequent embodiment, wherein deprotection is achieved by hydrogenation.

[0178] A composition according to any preceding or subsequent embodiment, wherein hydrogenation is carried out in the presence of a Pd / C catalyst.

[0179] A composition according to any preceding or succeeding embodiment, wherein hydrogenation is carried out in the presence of a Pd / Si catalyst.

[0180] The composition according to any preceding or succeeding embodiment, wherein hydrogenation is carried out at a temperature of about 10-40°C, or about 20-30°C, or about 25°C.

[0181] A composition according to any preceding or succeeding embodiment, wherein hydrogenation is carried out at a pressure of about 0 to about 6 bar.

[0182] The composition according to any preceding or succeeding embodiment, wherein hydrogenation is carried out using at least one solvent selected from the group consisting of water, ethyl acetate, isopropyl acetate, methanol, ethanol, isopropanol, or mixtures thereof.

[0183] A composition according to any preceding or succeeding embodiment, on an anhydrous basis, comprising at least 90 wt%, for example 91 wt%, 92 wt%, 93 wt%, 94 wt%, 95 wt%, 96 wt%, and 97 wt%, of the compound of formula (I) or its stereoisomers, hydrates, or pharmaceutically acceptable salts thereof, as described above.

[0184] A composition according to any preceding or succeeding embodiment, on an anhydrous basis, comprising at least 90 wt%, for example 91 wt%, 92 wt%, 93 wt%, 94 wt%, 95 wt%, 96 wt%, and 97 wt%, of the compound of formula (Ia) or its hydrate or a pharmaceutically acceptable salt thereof, as described above.

[0185] A composition comprising, on an anhydrous basis, at least 90 wt%, for example, 91 wt%, 92 wt%, 93 wt%, 94 wt%, 95 wt%, 96 wt%, and 97 wt%, of the compound of formula (I) or its stereoisomers, hydrates, or pharmaceutically acceptable salts.

[0186] A composition containing, on an anhydrous basis, at least 90 wt%, such as 91 wt%, 92 wt%, 93 wt%, 94 wt%, 95 wt%, 96 wt%, and 97 wt% of a compound of formula (Ia) or a hydrate or pharmaceutically acceptable salt thereof.

[0187] A compound of formula (I)

Chem.

Chem.

Chem.

[0188] A compound of formula (Ia)

Chem.

Chem.

[0189] Compound of formula (I) [ka] or its stereoisomers, hydrates, or pharmaceutically acceptable salts, and compounds of formula (II). [ka] or its stereoisomers, hydrates, or pharmaceutically acceptable salts, and / or compounds of formula (III). [ka] or its stereoisomers, hydrates, or pharmaceutically acceptable salts, wherein R1, R2, R3, and R4 are independently hydrogen and C 1-4 Selected from the group consisting of alkyl groups, However, if at least one of R1, R2, R3, and R4 is C 1-4 It is alkyl, A composition that contains, on an anhydrous basis, at least 90 wt%, such as 91 wt%, 92 wt%, 93 wt%, 94 wt%, 95 wt%, 96 wt%, and 97 wt% of a compound of formula (I).

[0190] A compound of formula (Ia)

Chem.

Chem.

Chem.

[0191] The following steps a) Coupling H-MePro-OH and Z-Gly-OH in the presence of an activator, a silylating agent, and a solvent, or b) Coupling Z-Gly-OH and Suc-OH in the absence or presence of an activator and in the absence or presence of a silylating agent, and then coupling the resulting Z-Gly-OSu and H-MePro-OH. c) Coupling the obtained Z-Gly-MePro-OH with H-Glu-OH in the presence of an activator, a silylation agent, and a solvent, d) To obtain Z-Gly-MePro-Glu-OH, e) A method for producing a composition containing the trofinetide according to any preceding or subsequent embodiment, comprising the steps of producing, wherein the trofinetide is dissolved or suspended in a solvent.

[0192] A method of any preceding or succeeding embodiment in which the silylating agent does not contain a cyano group.

[0193] A method of any preceding or succeeding embodiment, wherein the silylation agent is selected from the group consisting of N,O-bis(trimethylsilyl)acetamide, N,O-bis(trimethylsilyl)trifluoroacetamide, hexamethyldisilazane, N-methyl-N-(trimethylsilyl)acetamide (TMA), N-methyl-N-(trimethylsilyl)trifluoroacetamide, N-(trimethylsilyl)acetamide, N-(trimethylsilyl)diethylamine, N-(trimethylsilyl)dimethylamine, 1-(trimethylsilyl)imidazole, and 3-(trimethylsilyl)2-oxazolidone.

[0194] A method of any preceding or succeeding embodiment, wherein the solvent in step a) is a polar organic solvent or a polar aprotic organic solvent.

[0195] The method of any preceding or succeeding embodiment, wherein the solvent in step b) is a polar organic solvent or a polar aprotic organic solvent.

[0196] The method of any preceding or succeeding embodiment, wherein the solvent in step c) is a polar organic solvent or a polar aprotic organic solvent.

[0197] The method of any preceding or subsequent embodiment, wherein the solvent is selected from the group consisting of alkyl acetate, chlorinated hydrocarbon, alkyl cyanide, and amide solvents, or a mixture thereof.

[0198] The method of any preceding or subsequent embodiment, wherein the activator is selected from the group consisting of carbodiimide, acyl halide, phosphonium salt, uronium salt and guanidinium salt.

[0199] The method of any preceding or subsequent embodiment, wherein deprotection is achieved by hydrogenation.

[0200] The method of any preceding or subsequent embodiment, wherein hydrogenation is carried out in the presence of a Pd / C catalyst.

[0201] The method of any preceding or subsequent embodiment, wherein hydrogenation is carried out in the presence of a Pd / Si catalyst.

[0202] The method of any preceding or subsequent embodiment, wherein hydrogenation is carried out at a temperature of about 10 - 40 °C, or about 20 - 30 °C, or about 25 °C.

[0203] The method of any preceding or subsequent embodiment, wherein hydrogenation is carried out at a pressure of about 0 - about 6 bar.

[0204] The method of any preceding or subsequent embodiment, wherein hydrogenation is carried out in at least one solvent selected from the group consisting of water, ethyl acetate, isopropyl acetate, methanol, ethanol, isopropanol, or a mixture thereof.

[0205] A product comprising the composition according to any preceding or subsequent embodiment.

[0206] The product according to any preceding or subsequent embodiment, wherein the product is a kit.

[0207] Compound of formula (Ia)

Chemical formula

[0208] A kit of any preceding or succeeding embodiment further comprising one or more compounds selected from the group consisting of formulas (III), (IIIa), (IV), (V), (VI), (VII), (VIII), and (IX).

[0209] Compound of formula (Ia) [ka] or its hydrate, or a pharmaceutically acceptable salt thereof, and the compound of formula (IIa). [ka] A product of any preceding or subsequent embodiment, comprising a kit comprising a dosage form comprising or a hydrate or pharmaceutically acceptable salt thereof, and equipped with instructions for use, wherein the product comprises a specified amount of the compound of formula (Ia) in the product between 95 wt% and 105 wt%, for example, 96 wt%, 97 wt%, 98 wt%, 99 wt%, 100 wt%, 101 wt%, 102 wt%, 103 wt%, or 104 wt%.

[0210] Compound of formula (Ia) [ka] or its hydrate, or a pharmaceutically acceptable salt thereof, and the compound of formula (IIa). [ka] A product of any prior embodiment comprising a kit comprising a dosage form with instructions for use, further comprising one or more compounds selected from the group consisting of formulas (III), (IIIa), (IV), (V), (VI), (VII), (VIII), and (IX), wherein the composition comprises between 95 wt% and 105 wt% of a specified amount of the compound of formula (Ia) in the product, for example, 96 wt%, 97 wt%, 98 wt%, 99 wt%, 100 wt%, 101 wt%, 102 wt%, 103 wt%, or 104 wt%.

[0211] Compound of formula (Ia) [ka] or its hydrate, or a pharmaceutically acceptable salt thereof, and the compound of formula (IIa). [ka] A product of any preceding or subsequent embodiment, comprising a kit comprising a dosage form comprising or a hydrate or pharmaceutically acceptable salt thereof, and equipped with instructions for use, wherein the product comprises a specified amount of the compound of formula (Ia) in the product between 95 wt% and 105 wt%, for example, 96 wt%, 97 wt%, 98 wt%, 99 wt%, 100 wt%, 101 wt%, 102 wt%, 103 wt%, or 104 wt%.

[0212] Compound of formula (Ia) [ka] or its hydrate, or a pharmaceutically acceptable salt thereof, and the compound of formula (IIa). [ka] A product of any prior embodiment comprising a kit comprising a dosage form with instructions for use, further comprising one or more compounds selected from the group consisting of formulas (III), (IIIa), (IV), (V), (VI), (VII), (VIII), and (IX), wherein the composition comprises between 95 wt% and 105 wt% of a specified amount of the compound of formula (Ia) in the product, for example, 96 wt%, 97 wt%, 98 wt%, 99 wt%, 100 wt%, 101 wt%, 102 wt%, 103 wt%, or 104 wt%.

[0213] Preparation of compounds and compositions of formula (I) The preparation of the compounds of this disclosure produces compounds having stereochemistry defined by the stereochemistry of the reactants, and this will be well understood by those skilled in the art. Where the reactants have mixed stereochemistry, for example, a racemate or other variation of its stereoisomer, the resulting compounds will have mixed stereochemistry accordingly. In certain embodiments and aspects relating to the compounds and compositions disclosed herein, stereochemistry is shown, but in other cases it is not. Whenever a formula or chemical structure does not refer to the stereochemistry of a compound and / or composition, it should be understood to relate to all conceivable stereochemical aspects.

[0214] It should be understood that the process described herein is only a specific step. Other schemes can be developed to produce the compound of formula I.

[0215] Manufacturing of trophinetide (formula (Ia)) A method for producing a composition containing trophinetide generally involves the following steps: a) Coupling H-MePro-OH with Z-Gly-OH in the presence of an activator, a silylation agent, and a solvent, or b) Coupling Z-Gly-OH with Suc-OH in the absence or presence of an activator and solvent, and in the absence or presence of a silylating agent, and then coupling the resulting Z-Gly-OSu with H-MePro-OH, c) Coupling the obtained Z-Gly-MePro-OH with H-Glu-OH in the presence of an activator, a silylation agent, and a solvent, d) Obtain Z-Gly-MePro-Glu-OH with a conversion rate of at least 95%, e) Deprotecting Z-Gly-MePro-Glu-OH, wherein deprotection involves dissolving or suspending Z-Gly-MePro-Glu-OH in a solvent to produce a trophinetide.

[0216] These general steps will be explained in detail in the following examples.

[0217] Manufacturing of other compounds Certain other products can be manufactured together with the compound of formula (I) and may be present in the compositions disclosed herein. An example of the compound of formula (I) or its stereoisomer is formula (Ia) (tropinetide). Other products include the compounds (II), (IIa), (III), (IIIa), (IV), (V), (VI), (VII), (VIII), and (IX) described above.

[0218] The additional products include dosage forms containing the compound of formula (Ia) (trophinetide) and the compound of formula (IIa), as well as a kit containing instructions for use.

[0219] The kit further comprises one or more compounds selected from the group consisting of formulas (III), (IIIa), (IV), (V), (VI), (VII), (VIII), and (IX). [Examples]

[0220] The preparation of compositions containing trofinetide is illustrated below. It can be understood that other schemes may be used to prepare trofinetide.

[0221] Example 1: Trophynetide Manufacturing Process

[0222] Generally, trophinetides and related compounds can be produced from precursor peptides or amino acids reacted with a silylating agent or persilylating agent in one or more steps. In the present invention, silylating agents such as N-trialkylsilylamines or N-trialkylsilylamides that do not contain a cyano group can be used.

[0223] Examples of such silylating agents include N,O-bis(trimethylsilyl)acetamide (BSA), N,O-bis(trimethylsilyl)trifluoroacetamide, hexamethyldisilazane, N-methyl-N-(trimethylsilyl)acetamide (TMA), N-methyl-N-(trimethylsilyl)trifluoroacetamide, N-(trimethylsilyl)acetamide, N-(trimethylsilyl)diethylamine, N-(trimethylsilyl)dimethylamine, 1-(trimethylsilyl)imidazole, and 3-(trimethylsilyl)2-oxazolidone.

[0224] Step 1: Preparation of Z-Gly-OSu Several alternative procedures can be used in this process.

[0225] Step 1A [ka] One equivalent of Z-Gly-OH and 1.1 equivalents of Suc-OH were solubilized in 27 equivalents of iPrOH and 4 equivalents of CH2Cl2 at 21°C. The mixture was cooled, and when the temperature reached -4°C, 1.1 equivalents of EDC.HCl were gradually added, while maintaining the temperature below 10°C. During the reaction, a dense solid appeared. After adding EDC.HCl, the mixture was warmed to 20°C. The suspension was cooled to 11°C and filtered. The mass was washed with 4.9 equivalents of cold iPrOH and 11 equivalents of IPE, and then dried at 34°C (Z-Gly-OSu dried product - purity: 99.5%, NMR assay: 96%, yield: 84%).

[0226] Step 1B This procedure is a variation of procedure 1A, differing in that iPrOH is replaced with ACN. 1 equivalent of Z-Gly-OH and 1.1 equivalents of Suc-OH were solubilized in 22 equivalents of ACN at 35°C. The mixture was cooled in an ice bath. Once the temperature reached 1°C, 0.9 equivalents of DCC were gradually added to 5.5 equivalents of ACN, maintaining the temperature below 5°C. The coupling reaction took approximately 20 hours. During the reaction, DCU precipitated and was removed by filtration at the end of the coupling. After filtration, DCU was washed with ACN to recover the product. Next, the Z-Gly-OSu mixture was concentrated to 60% by weight. 17 equivalents of iPrOH were added to initiate crystallization. A dense solid appeared immediately after the addition of iPrOH. An additional 17 equivalents of iPrOH were needed to liquefy the suspension. This suspension was cooled in an ice bath and filtered. The solid was washed with 9 equivalents of iPrOH and then dried at 45°C (Z-Gly-OSu dried product - purity: 99.2%, HPLC assay: 99.6%, yield: 71%).

[0227] Step 2: Preparation of Z-Gly-MePro-OH Several alternative procedures can be used in this process.

[0228] Step 2A [ka] One equivalent of MePro.HCl was partially solubilized at 35°C in 29 equivalents of CH2Cl2 with 1.04 equivalents of TEA and 1.6 equivalents of TMA. The mixture was heated at 35°C for 2 hours to silylate it. Next, 1.02 equivalents of Z-Gly-OSu were added to the mixture. The mixture was maintained at 35°C for 3 hours, and then 0.075 equivalents of butylamine were added to quench the reaction. The mixture was allowed to return to room temperature and mixed for at least 15 minutes. Z-Gly-MePro-OH was extracted once with 5% w / w NaHCO3 in 186 equivalents of water, and then three consecutively with 5% w / w NaHCO3 in 62 equivalents of water. The aqueous layer was pooled and the pH was adjusted to 2.2 by adding 34 equivalents of HCl as 12N HCl at room temperature. At this pH, Z-Gly-MePro-OH formed a viscous solid and was solubilized at 45°C with approximately 33 equivalents of pharmaceutically acceptable ammonium compounds and 2.3 equivalents of iButOH. Z-Gly-MePro-OH was extracted into the organic layer and washed with 62 equivalents of desalted water. The organic layer was then dried by azeotropic distillation with 11.5 equivalents of pharmaceutically acceptable ammonium compounds until the peptide began to precipitate. Cyclohexane (12 equivalents) was added to the mixture to complete the precipitation. The suspension was cooled at 5°C for 2 hours and filtered. The solid was washed with 10 equivalents of cyclohexane and dried at 45°C (Z-Gly-MePro-OH dried product - purity: 100%, HPLC assay: 100%, yield 79%).

[0229] Step 2B This procedure is a variation of procedure 2A. One equivalent of MePro.HCl was partially solubilized at 34°C in 36.6 equivalents of CH2Cl2 with 1.01 equivalents of TEA and 0.1 equivalents of TMA. Next, 1.05 equivalents of Z-Gly-OSu were added to the mixture, followed by 1.0 equivalent of TEA. The mixture was maintained at 35°C for about 1 hour, cooled to 25-30°C, and the reaction was stopped by adding 0.075 equivalents of DMAPA. 100 equivalents of water, 8.6 equivalents of HCl as 12N HCl, and 0.3 equivalents of KHSO4 were added to the mixture (no precipitate was observed, pH=1.7). Z-Gly-MePro-OH was extracted into the organic layer and washed twice with 97 equivalents of deionized water and 0.3 equivalents of KHSO4, respectively, followed by washing with 100 equivalents of deionized water. 23 equivalents of siRNA were added to the mixture, and CH2Cl2 was removed by distillation until the peptide began to precipitate. 25 equivalents of cyclohexane were added to the mixture to complete the precipitation. The suspension was cooled overnight at -2°C and filtered. The solid was washed with 21 equivalents of cyclohexane and then dried at 39°C (Z-Gly-MePro-OH dried product - purity: 98.7%, NMR assay: 98%, yield 86%).

[0230] Step 2C [ka] In reactor 1, MePro.HCl (1 equivalent) was suspended in SiO (approximately 7 equivalents). DIPEA (1 equivalent) and TMA (2 equivalents) were added, and the mixture was heated to dissolve the solid. After dissolution, the solution was cooled to 0°C. In reactor 2, Z-Gly-OH (1 equivalent) was suspended in SiO (approximately 15 equivalents). DIPEA (1 equivalent) and pyridine (1 equivalent) were added. After mixing, a solution was obtained and cooled to -5°C. Piv-Cl (1 equivalent) was added to reactor 2, and the contents of reactor 1 were added to reactor 2. After the addition was complete, the contents of reactor 2 were allowed to rise to room temperature. The conversion from Z-Gly-OH to Z-Gly-MePro-OH was monitored by HPLC. After the reaction was complete, the reaction mixture was quenched with DMAPA (0.1 equivalent) and washed with an aqueous solution consisting of KHSO4 (approx. 2.5 wt%), NaCl (approx. 4 wt%), and concentrated HCl (approx. 6 wt%) in 100 equivalents of H2O. The aqueous layer was re-extracted with ethyl acetate, and the combined organic layer was washed with an aqueous solution consisting of KHSO4 (approx. 2.5 wt%) and NaCl (approx. 2.5 wt%) in 100 equivalents of H2O, and then with water (100 equivalents). The residual water was removed from the organic solution of Z-Gly-MePro-OH by vacuum distillation using ethyl acetate. The resulting suspension was diluted with heptane (approx. 15 equivalents) and cooled to 0°C. The product was isolated by filtration, washed with cold heptane (approx. 7 equivalents), and dried under vacuum at 45°C. Z-Gly-MePro-OH (85% yield) was obtained.

[0231] Step 3: Preparation of Z-Gly-MePro-Glu-OH Several alternative procedures can be used in this process.

[0232] Step 3A [ka] H-Glu-OH (1.05 equivalents) was silylated at 65°C with 3.5 equivalents of TMA and 2 equivalents of CH2Cl2. Silylation was completed after 2 hours. While silylation was in progress, 1.0 equivalent of Z-Gly-MePro-OH and 1.0 equivalent of Oximapure were solubilized in a separate reactor at room temperature with 24 equivalents of CH2Cl2 and 1.0 equivalent of DMA. EDC.HCl (1.0 equivalent) was added. The activation rate reached 97% after 15 minutes. Next, the activated Oximapure solution was added to the silylated H-Glu-OH at 40°C and cooled to room temperature. The coupling time was approximately 15 minutes, and the coupling rate was 97%. The pH was adjusted to 8 by adding a mixture of 8.2% w / w NaHCO3 in 156 equivalents of water at room temperature (releasing CO2). Z-Gly-MePro-Glu-OH was extracted in water. The aqueous layer was washed twice with 29 equivalents of CH2Cl2. Residual CH2Cl2 was removed by concentration. After adjusting the pH to 2.5 with 2.5N HCl, 1.4 equivalents of solid KHSO4 were added to precipitate Z-Gly-MePro-Glu-OH. The mixture was filtered, and the solid was washed three times with 52 equivalents of water. The filtered solid was added to 311 equivalents of desalted water and heated to 55-60°C. iPrOH (29 equivalents) was gradually added until the product was completely solubilized. The mixture was slowly cooled to 10°C for 40 minutes under moderate mixing to initiate crystallization. The peptide was filtered, washed twice with 52 equivalents of water, and then dried at 45°C (Z-Gly-MePro-Glu-OH dried product - purity: 99.5%, NMR assay: 96%, yield 74%).

[0233] Step 3B One equivalent of Z-Gly-MePro-OH and 1.05 equivalents of Suc-OH were solubilized in 40 equivalents of ACN and 30 equivalents of CH2Cl2 at room temperature. The mixture was cooled in an ice bath, and as the temperature approached 0°C, 1.05 equivalents of DCC dissolved in 8 equivalents of ACN were gradually added while maintaining the temperature below 5°C. After adding DCC, the mixture was gradually heated from 0°C to 5°C over 1 hour, then to 20°C over 1-2 hours, and then to 45°C over 2-5 hours. After 5 hours, the mixture was cooled to 5°C and maintained overnight. The activation rate reached 98% after approximately 24 hours. DCU was removed by filtration and washed with 13.5 equivalents of ACN. During the activation process, 1.1 equivalents of H-Glu-OH were silylated at 65°C with 30 equivalents of ACN and 2.64 equivalents of TMA. Silylation was completed after 2 hours. Z-Gly-MePro-OSu was then gradually added to silylated H-Glu-OH at room temperature, and 0.4 equivalents of TMA were added to maintain the solubility of H-Glu-OH. The mixture was heated to 45°C, and if precipitation occurred, 0.7 equivalents of TMA were added. The coupling time was approximately 24 hours, and the coupling rate was approximately 91%. The reaction was quenched by adding 0.15 equivalents of butylamine and 2.0 equivalents of TEA. Water (233 equivalents) was added, and the mixture was concentrated until gelation occurred. Z-Gly-MePro-Glu-OH was extracted into water by adding 233 equivalents of water and 5% w / w NaHCO3 in 132 equivalents of CH2Cl2. The aqueous layer was washed twice with 44 equivalents of CH2Cl2. Residual CH2Cl2 was removed by distillation. The pH was adjusted to 2.0 with 24 equivalents of HCl as 12N HCl, followed by 75 equivalents of HCl as 4N HCl. At this pH, Z-Gly-MePro-Glu-OH precipitated. The mixture was cooled in an ice bath for more than 1 hour and filtered. The solid was washed with 186 equivalents of cold water and then dried at 45°C (Z-Gly-MePro-Glu-OH dried product - HPLC purity: 98.4%, NMR assay: 100%, yield 55%).

[0234] Step 3C This procedure is a variation of procedure 3A. 1.05 equivalents of H-Glu-OH were silylated with 3.7 equivalents of CH2Cl2 and 3.5 equivalents of TMA at 62°C. Silylation was completed in approximately 1.5–2 hours, as evidenced by solubilization. During the silylation process, 1.0 equivalent of Z-Gly-MePro-OH and 1.0 equivalent of Oxymapure were solubilized in 31.5 equivalents of CH2Cl2 at 22°C. Activation was completed by adding 1 (1.06) equivalents of EDC.HCl. Next, the silylated H-Glu-OH was added to the activated Oxymapure solution. During the addition, the temperature was controlled to remain below 45°C. Desilylation was performed by adding a mixture of 2.5% w / w KHSO4 and 9 equivalents of iPrOH in 153 equivalents of water, resulting in a pH of 1.65. Residual CH2Cl2 was removed by concentration. The mixture was cooled to 12°C to precipitate Z-Gly-MePro-Glu-OH. The mixture was filtered, the solid was washed with 90 equivalents of water, and then dried at 36°C.

[0235] Step 3D This procedure is a variation of procedure 3A. 1.05 equivalents of H-Glu-OH were silylated with 3.9 equivalents of CH2Cl2 and 3.5 equivalents of TMA at 62°C. Silylation was completed after 2 hours, as evidenced by solubilization. During the silylation process, 1.0 equivalent of Z-Gly-MePro-OH and 1.0 equivalent of Oximapure were solubilized in 25 equivalents of CH2Cl2 at 23°C. 1 equivalent of EDC and HCl were added. 0.07 equivalents of EDC were added to complete the activation. HCl was added. Next, the silylated H-Glu-OH was added to the activated Oximapure solution. During the addition, the temperature was controlled to remain below 45°C. Desilylation was performed by adding a mixture of 2.5% w / w KHSO4 and 9.6 equivalents of iPrOH in 160 equivalents of water, resulting in a pH of 1.63.

[0236] Residual CH2Cl2 was removed by concentration. The mixture was cooled to 20°C to precipitate Z-Gly-MePro-Glu-OH. The mixture was filtered, and the solid was washed with 192 equivalents of water and dried at 25°C for 2.5 days. The solid was then solubilized at 64°C by adding 55 equivalents of water and 31 equivalents of iPrOH. After solubilization, the mixture was diluted with 275 equivalents of water and cooled to 10°C for crystallization. The mixture was filtered, washed with 60 equivalents of water, and dried at 27°C (Z-Gly-MePro-Glu-OH dried product - purity: 99.6%, NMR assay: 98%, yield 74%).

[0237] Step 3E [ka] In reactor 1, H-Glu-OH (1.05 equivalents) was suspended in ACN (approximately 2.2 equivalents). TMA (approximately 3.5 equivalents) was added, and the mixture was heated to dissolve the solid. After dissolution, the solution was cooled to room temperature. In reactor 2, Z-Gly-MePro-OH (1 equivalent) was suspended in ACN (14 equivalents). Oxymapure (1 equivalent) and EDC.HCl (1 equivalent) were added. The mixture was stirred at room temperature until the solid dissolved. The contents of reactor 2 were added to reactor 1. The conversion from Z-Gly-MePro-OH to Z-Gly-MePro-Glu-OH was monitored by HPLC. Once complete, the reaction mixture was added to an aqueous solution consisting of KHSO4 (approximately 2.5 wt%) dissolved in approximately 100 equivalents of H2O. ACN was removed from the aqueous suspension of Z-Gly-MePro-Glu-OH by vacuum distillation using H2O. After stirring at room temperature, the product in the resulting suspension was isolated by filtration and washed with water. The obtained solid was dissolved in an aqueous solution of NaHCO3 (approximately 5 wt%) in 110 equivalents of H2O, and recrystallized by adding an aqueous solution of KHSO4 (approximately 10 wt%) in 90 equivalents of H2O. The product was isolated by filtration, washed with water, and dried under vacuum at 45°C. Z-Gly-MePro-Glu-OH (75% yield) was obtained.

[0238] Step 4: Deprotection and isolation of trophinetide Several alternative procedures can be used in this process.

[0239] Step 4A [ka] Z-Gly-MePro-Glu-OH (1 equivalent) was suspended in water (approximately 25 equivalents) and ethyl acetate (approximately 15 equivalents). Pd / C (containing 10% by weight of Pd at 0.025 weight equivalents) was added, and the reaction mixture was hydrogenated by bubbling hydrogen into the reaction mixture at room temperature. The conversion of Z-Gly-MePro-Glu-OH to trofinetide was monitored by HPLC, and once the reaction was complete, the catalyst was removed by filtration and the layers were separated. Residual ethyl acetate was removed from the aqueous solution containing trofinetide by sparging with nitrogen or washing with heptane. The aqueous solution was spray-dried to isolate the product. Trophinetide (90% yield) was obtained. Alternatively, deprotection can be achieved using MeOH alone, or a combination of iPrOH and MeOH, or by using ethyl acetate in water.

[0240] Step 4B This procedure is a variation of procedure 4A, excluding RINKAN. Z-Gly-MePro-Glu-OH (1 equivalent) was suspended in water (approximately 50 equivalents). Pd / C (0.05 equivalents, 5 wt% Pd) was added, and the reaction mixture was hydrogenated at room temperature under a pressure of 5 bar. The conversion of Z-Gly-MePro-Glu-OH to trofinetide was monitored by HPLC. Once the reaction was complete, the catalyst was removed by filtration, and the aqueous layer was washed with RINKAN (approximately 5 equivalents). Residual RINKAN was removed from the aqueous solution containing trofinetide by sparging with nitrogen or washing with heptane. The aqueous solution was spray-dried to isolate the product. Trophinetide (90% yield) was obtained.

[0241] Step 4C This procedure is a variation of procedure 4A, replacing siRNA with MeOH. Z-Gly-MePro-Glu-OH (1 equivalent) was suspended in MeOH (100 equivalents) and water (12 equivalents). Pd / Si (0.02 equivalents by weight) was added, and the mixture was heated at 23°C for hydrogenolysis. Solubilization of the peptide occurred during deprotection. The conversion of Z-Gly-MePro-Glu-OH to trofinetide was monitored by HPLC, and once the reaction was complete, the catalyst was removed by filtration, and the layers were washed with MeOH and iPrOH. The solvent was concentrated under vacuum at 45°C to precipitate the trofinetide. The precipitate was filtered and dried at 45°C to obtain trofinetide.

[0242] Step 4D This procedure is a variation of procedure 4A, replacing Pd / C with Pd / Si. One equivalent of Z-Gly-MePro-Glu-OH was partially solubilized in 105 equivalents of MeOH and 12 equivalents of water. Pd / Si (0.02 equivalents by weight) was added, and the mixture was heated at 23°C for hydrocracking. Solubilization of the peptide occurred during deprotection. At the end of deprotection (conversion rate approximately 99% after 1 hour), the catalyst was filtered off and washed with 20-30 equivalents of MeOH. MeOH was replaced with iPrOH by adding iPrOH (93 equivalents) and concentrating at 45°C under vacuum. The peptide was concentrated until it began to precipitate. The peptide was filtered and dried at 45°C (H-Gly-MePro-Glu-OH dried product: purity: 98.1%, NMR assay: 90%, yield 81%).

[0243] Step 4E This procedure is a variation of procedure 4A, removing H2O and replacing Pd / C with Pd / Si. One equivalent of Z-Gly-MePro-Glu-OH was partially solubilized in 44 equivalents of MeOH. Pd / Si type 340 (0.02 equivalents by weight) was added, and the mixture was kept at 20°C for hydrogenolysis. Solubilization of the peptide occurred during deprotection. At the end of deprotection (conversion rate approximately 99.9%, after 3-3.5 hours), the catalyst was filtered off and washed with 8 equivalents of MeOH. The deprotected peptide was then precipitated in 56 equivalents of iPrOH. After 30 minutes at 5°C, the peptide was filtered, washed three times with 11 equivalents of iPrOH, and then dried at 25°C (H-Gly-MePro-Glu-OH dried product: purity: 99.4%, HPLC assay: approximately 98%, yield: 81%).

[0244] Step 4F This procedure is a variation of procedure 4A. One equivalent of Z-Gly-MePro-Glu-OH was partially solubilized in 14 equivalents of ethyl acetate and 25 equivalents of water. Pd / C (0.01 equivalents by weight) was added, and the mixture was kept at 20°C for hydrocracking. Solubilization of the peptide occurred during deprotection. At the end of deprotection (conversion rate approximately 100%, after approximately 3.5 hours), the catalyst was filtered off and washed with a mixture of 3.5 equivalents of ethyl acetate and 6 equivalents of water. The aqueous layer was then ready for spray drying (H-Gly-MePro-Glu-OH peptide aqueous solution: purity: 98.6%, yield: approximately 95%).

[0245] Step 4G This procedure is a variation of procedure 4A, with the substitution of Pd / C and Pd / Si, the substitution of siRNA and MeOH, and the removal of H2O. Pd / Si type 340 (0.02 equivalents by weight) was added to 2.9 volumes of MeOH, and a preliminary reduction was carried out for 30 minutes. 1 equivalent of Z-Gly-MePro-Glu-OH was partially solubilized in 34 equivalents of MeOH. Next, the reduced palladium was transferred to the peptide mixture. The mixture was kept at 20°C for hydrogenolysis. Solubilization of the peptide occurred during deprotection. Pd / C type 39 (0.007 equivalents by weight) was added to the mixture to increase the reaction rate. At the end of deprotection, the catalyst was filtered off and washed with 13.6 equivalents of MeOH. Next, the deprotected peptide was precipitated in 71 equivalents of iPrOH. After about 40 minutes, the peptide was filtered and washed with 35 equivalents of iPrOH. After drying the peptide at a temperature below 20°C, it was solubilized in water and ready for spray drying.

[0246] Step 4H This procedure is a variation of procedure 4A. One equivalent of Z-Gly-MePro-Glu-OH was partially solubilized in 24.8 equivalents of water and 13.6 equivalents of siRNA. Pd / C type 39 (0.025 equivalents by weight) was added to the peptide mixture. The mixture was kept at 20°C for hydrocracking. Solubilization of the peptide occurred during deprotection. At the end of deprotection (19 hours), the catalyst was removed by filtration and washed with 5.3 equivalents of water and 2.9 equivalents of siRNA. The two-phase mixture was then decanted to remove the upper organic layer. The aqueous layer was diluted with water to an H-Gly-MePro-Glu-OH concentration suitable for spray-drying the solution.

[0247] Example 2: Alternative Trophynetide Manufacturing Process An alternative method for synthesizing trofinetide is based on U.S. Patent No. 8,546,530, which is suitable for tripeptides, as follows:

[0248] The persilylated compounds used to synthesize formula (Ia) (trophinetide) are obtained by silylation of the corresponding peptide or amino acid by reaction with a silylating agent, optionally in an organic solvent. The persilylated peptide or amino acid can be isolated and purified, optionally. For example, the persilylated peptide or amino acid can be used in situ by combining a solution containing the persilylated peptide or amino acid with a solution containing the optionally activated peptide or amino acid.

[0249] In step 2, the amino acid persilylated compound is obtained by silylation of the corresponding amino acid (e.g., H-MePro-OH) by reaction with a silylating agent in an organic solvent, if desired. The persilylated amino acid can be isolated and purified if desired. For example, the persilylated amino acid can be used in situ by combining a solution containing the persilylated amino acid with a solution containing an activated amino acid (e.g., Z-Gly-OH), if desired.

[0250] In step 3, the amino acid persilylated compound is obtained by silylation of the corresponding amino acid (e.g., H-MePro-OH) by reaction with a silylating agent, optionally in an organic solvent. The persilylated amino acid or peptide can be isolated and purified, optionally. It is useful for in situ use of the persilylated amino acid or peptide by combining, for example, a solution containing the persilylated amino acid with a solution containing a peptide (e.g., Z-Gly-MePro-OH), optionally activated (e.g., by using EDC.HCl and OximaPure).

[0251] In the present invention, it is useful to use silylation agents that do not contain a cyano group, such as N-trialkylsilylamines or N-trialkylsilylamides. Examples of such silylation agents include N,O-bis(trimethylsilyl)acetamide (BSA), N,O-bis(trimethylsilyl)trifluoroacetamide, hexamethyldisilazane, N-methyl-N-(trimethylsilyl)acetamide (TMA), N-methyl-N-(trimethylsilyl)trifluoroacetamide, N-(trimethylsilyl)acetamide, N-(trimethylsilyl)diethylamine, N-(trimethylsilyl)dimethylamine, 1-(trimethylsilyl)imidazole, and 3-(trimethylsilyl)2-oxazolidone.

[0252] The reaction in step 2 is generally carried out at temperatures of 0°C to 100°C, preferably 10°C to 40°C, and preferably 15°C to 30°C.

[0253] The reaction in step 3 is generally carried out at temperatures of 0°C to 100°C, preferably 10°C to 60°C, and preferably 15°C to 50°C.

[0254] In the reaction of step 2, generally 0.5 to 5 equivalents, preferably 1 to 3 equivalents, or preferably about 1.5 to 2.5 equivalents of silylating agent are used relative to the molar amount of the functional group to be silylated. It is also possible to use 2 to 4 equivalents of silylating agent relative to the molar amount of the functional group to be silylated. The term "functional group to be silylated" specifically refers to a group having an active hydrogen atom that reacts with the silylating agent, such as an amino group, hydroxyl group, mercapto group, or carboxyl group.

[0255] In the reaction of step 3, generally 0.5 to 5 equivalents of silylating agent are used relative to the molar amount of the functional group to be silylated, preferably 2 to 4.5 equivalents, or preferably about 3 to 4 equivalents. It is also possible to use 2.5 to 4.5 equivalents of silylating agent relative to the molar amount of the functional group to be silylated.

[0256] "Persilylation" is understood to mean an amino acid or peptide, or an amino acid analog or peptide analog, that is sufficiently silylated to ensure that a group having an active hydrogen atom that can react with a silylation agent is obtained, thereby ensuring a homogeneous reaction medium for the coupling step.

[0257] In the process according to the present invention, the reaction between an amino acid or peptide and a persilylated amino acid or peptide is often carried out in the presence of a carboxyl group activator. In this case, the carboxylic acid activator is appropriately selected from carbodiimides, acyl halides, phosphonium salts, and uronium or guanidinium salts. More optionally, the carboxylic acid activator is an acyl halide such as isobutyl chloroformate or pivaloyl chloride, or a carbodiimide such as EDC.HCl or DCC.

[0258] The use of additional carboxylic acid activators to reduce side reactions and / or increase reaction efficiency often yields better results. For example, phosphonium and uronium salts can convert protected amino acids to activated species in the presence of tertiary bases such as N,N-diisopropylethylamine (DIPEA) and triethylamine (TEA). Other reagents help prevent racemization by providing protective agents. These reagents include carbodiimides (e.g., DCC) or derivatives thereof that have been added with co-nucleophiles (e.g., 1-hydroxybenzotriazole (HOBt), 1-hydroxyazabenzotriazole (HOAt), or Suc-OH). Another available reagent is TBTU. Mixed anhydride methods using isobutyl chloroformate, with or without the addition of co-nucleophiles, are also used, similar to azide methods, due to the lower associated racemization. These types of compounds can also increase the rate of coupling via carbodiimides. Typical additional reagents may include bases such as N,N-diisopropylethylamine (DIPEA), triethylamine (TEA), or N-methylmorpholine (NMM).

[0259] When silylation is carried out in the presence of a solvent, the solvent is preferably a polar organic solvent, and more optionally a polar aprotic organic solvent. Amide solvents such as N,N-dimethylformamide (DMF) or N,N-dimethylacetamide (DMAC) can be used. In the present invention of step 2, an alkyl acetate solvent can be used, and ethyl acetate in particular is more specifically optional.

[0260] In step 3 of the present invention, a chlorinated hydrocarbon solvent or an alkyl cyanide solvent can be used, and more specifically, dichloromethane or acetonitrile are optional.

[0261] In another embodiment, silylation is carried out in a liquid silylation medium which essentially consists of a silylation agent and an amino acid or peptide.

[0262] In the present invention, an amino acid or peptide is understood to mean an amino acid or peptide, or an amino acid analog or peptide analog, in particular, an amino acid or peptide to which the carboxyl group of an amino-protected amino acid or peptide is bonded at its N-terminus or at any other position.

[0263] Example 3: Specifications of a composition containing the compound of formula (I) [Table 1] 1 ICH Guidelines Q3C on Impurities: Guidelines for Residual Solvents

[0264] Example 4: Example 1, Step 4, Procedure 4B of the production of a substitute for trofinetide

[0265] This procedure is a variation of Step 4, Step 4B. Z-Gly-MePro-Glu-OH (1 equivalent) was gradually added to approximately 50 equivalents of water containing Pd / C (0.027 equivalents by weight, 5% by weight of Pd). The reaction mixture was hydrogenated at 20°C at a pressure of 5 bar for at least 4 hours in 4 cycles. To accelerate the reaction, Pd / C (0.0027 equivalents by weight) was added between cycles as needed. The conversion of Z-Gly-MePro-Glu-OH to trofinetide was monitored by HPLC. Once the reaction was complete, the catalyst was removed by filtration and washed with water (12.5 equivalents), and the aqueous layer was washed with siRNA (approximately 14 equivalents). After phase separation, residual siRNA was removed from the aqueous solution containing trofinetide by sparging with nitrogen under vacuum for approximately 3 hours at 20°C. The aqueous solution was filtered. The final concentration of trophinetide was approximately 25 wt%, and the solution was then ready for spray drying to isolate the product.

[0266] Example 5: Alternative composition for trophinetide Compound of formula (I) [ka] or its stereoisomers, hydrates, or pharmaceutically acceptable salts, and compounds of formula (II). [ka] or its stereoisomers, hydrates, or pharmaceutically acceptable salts, and / or compounds of formula (III). [ka] A composition comprising either a stereoisomer thereof, a hydrate, or a pharmaceutically acceptable salt thereof, wherein R1, R2, R3, and R4 are independently hydrogen and C 1-4 Selected from the group consisting of alkyl groups, wherein at least one of R1, R2, R3, and R4 is C 1-4The composition is alkyl, and on an anhydrous basis, comprises at least 90 wt%, for example 91 wt%, 92 wt%, 93 wt%, 94 wt%, 95 wt%, 96 wt%, and 97 wt%, of the compound of formula (I).

[0267] Example 6: Alternative composition for trophinetide Compound of formula (Ia) [ka] or its hydrate, or a pharmaceutically acceptable salt thereof, and formula (II) [ka] or its stereoisomers, hydrates, or pharmaceutically acceptable salts, and / or compounds of formula (III). [ka] A composition comprising either a stereoisomer thereof, a hydrate, or a pharmaceutically acceptable salt thereof, wherein R1, R2, R3, and R4 are independently hydrogen and C 1-4 Selected from the group consisting of alkyl groups, wherein at least one of R1, R2, R3, and R4 is C 1-4 The composition is alkyl, and on an anhydrous basis, comprises at least 90 wt%, for example, 91 wt%, 92 wt%, 93 wt%, 94 wt%, 95 wt%, 96 wt%, and 97 wt%, of the compound of formula (Ia).

[0268] Example 7: Trophynetide product Compound of formula (Ia) [ka] or its hydrate, or a pharmaceutically acceptable salt thereof, and the compound of formula (IIa). [ka] A product comprising a kit comprising a dosage form comprising the hydrate or pharmaceutically acceptable salt thereof, and having instructions for use thereof, wherein the product comprises a specified amount of the compound of formula (Ia) in the product between 95 wt% and 105 wt%, for example, 96 wt%, 97 wt%, 98 wt%, 99 wt%, 100 wt%, 101 wt%, 102 wt%, 103 wt%, or 104 wt%.

[0269] Example 8: Trophynetide product Compound of formula (Ia) [ka] or its hydrate, or a pharmaceutically acceptable salt thereof, and the compound of formula (IIa). [ka] A product comprising a kit comprising a dosage form with instructions for use, further comprising one or more compounds selected from the group consisting of formulas (III), (IIIa), (IV), (V), (VI), (VII), (VIII), and (IX), wherein the composition comprises between 95 wt% and 105 wt% of a specified amount of the compound of formula (Ia) in the product, for example, 96 wt%, 97 wt%, 98 wt%, 99 wt%, 100 wt%, 101 wt%, 102 wt%, 103 wt%, or 104 wt%.

[0270] Example 9: Analysis of the product and composition Products and compositions disclosed herein can be analyzed using liquid chromatography, appropriate chromatographic methods using ULC, and materials and conditions such as Waters Acquity CSH C18, 1.7 μm, 150 x 2.1 mm column, water containing 0.1% TFA (mobile phase A), and water / ACN70 / 30 + 0.1% TFA (mobile phase B), (ranging from 4% A phase / 6% B phase to 100% B phase, flashed with 4% A phase / 6% B phase), flow rate: 0.35 ml / min, column temperature: 40°C, autosampler temperature: 4°C, and injection volume: 4 μl (for example, weighing approximately 10 mg of powder in a 10 ml volumetric flask and diluting with water). Examples of detectors include UV (ultraviolet, UV 220 nm) and MS (mass spectrometry).

[0271] [Industrial applicability] This invention finds use in the pharmaceutical, medical, and other healthcare fields. In certain embodiments, for example, the following items are provided: (Item 1) Compound of formula (I) [ka] or its stereoisomers, hydrates, or pharmaceutically acceptable salts, and compounds of formula (II). [ka] or its stereoisomers, hydrates, or pharmaceutically acceptable salts, and / or compounds of formula (III). [ka] A composition comprising either a stereoisomer thereof, a hydrate, or a pharmaceutically acceptable salt thereof, wherein R1, R2, R3, and R4 are independently hydrogen and C 1-4 Selected from the group consisting of alkyl groups, wherein at least one of R1, R2, R3, and R4 is C 1-4 The composition is alkyl. (Item 2) The compound according to formula (I) is the compound according to formula (Ia). [ka] The composition described in item 1, or its hydrate or a pharmaceutically acceptable salt thereof. (Item 3) The composition according to item 1 or 2, wherein the compound of formula (II), or its stereoisomers, hydrates, or pharmaceutically acceptable salts, is absent or present in an amount between approximately 0.001 ± 0.0002 wt% and approximately 2 ± 0.4 wt%, and the compound of formula (III), or its stereoisomers, hydrates, or pharmaceutically acceptable salts, is absent or present in an amount between approximately 0.001 ± 0.0002 wt% and approximately 2 ± 0.4 wt%, provided that at least one of the compounds of formula (II) or formula (III), or their stereoisomers, hydrates, or pharmaceutically acceptable salts, is present. (Item 4) A composition according to any one of items 1 to 3, wherein the compound of formula (II), or its stereoisomer, hydrate, or pharmaceutically acceptable salt is present in an amount between approximately 0.001 ± 0.0002 wt% and approximately 0.3 ± 0.06 wt%. (Item 5) A composition according to any one of items 1 to 4, wherein the compound of formula (II), or its stereoisomer, hydrate, or pharmaceutically acceptable salt is present in an amount between approximately 0.001 ± 0.0002 wt% and approximately 0.1 ± 0.02 wt%. (Item 6) The compound of formula (II), or its stereoisomer, hydrate, or pharmaceutically acceptable salt, is present in amounts between approximately 0.001±0.0002 wt% and approximately 2±0.4 wt%, or between approximately 0.001±0.0002 wt% and approximately 1.5±0.3 wt%, or between approximately 0.001±0.0002 wt% and approximately 1±0.2 wt%, or approximately 0.001±0. The composition according to item 1 or 2, present in an amount between 0.0002 wt% and about 0.7 ± 0.14 wt%, or in an amount between about 0.001 ± 0.0002 wt% and about 0.5 ± 0.1 wt%, or in an amount between about 0.001 ± 0.0002 wt% and about 0.3 ± 0.06 wt%, or in an amount between about 0.001 ± 0.0002 wt% and about 0.2 ± 0.04 wt%. (Item 7) The compound according to formula (II) is the compound according to formula (IIa). [ka] A composition according to any one of items 1 to 6, which is either a hydrate thereof or a pharmaceutically acceptable salt thereof. (Item 8) A composition according to any one of items 1 to 7, wherein the compound of formula (II), or its stereoisomer, hydrate, or pharmaceutically acceptable salt thereof, is not present. (Item 9) A composition according to any one of items 1 to 8, wherein the compound of formula (III), or its stereoisomer, hydrate, or pharmaceutically acceptable salt is present in an amount between about 0.001 ± 0.0002 wt% and about 2.0 ± 0.4 wt%. (Item 10) The composition comprises a compound of formula (III), and the compound of formula (III) is a compound of formula (IX). [ka] A composition according to any one of items 1 to 9, which is either a hydrate thereof or a pharmaceutically acceptable salt thereof. (Item 11) The compound comprises the compound of formula (III), wherein the compound of formula (III) is the compound of formula (IIIa). [ka] A composition according to any one of items 1 to 10, which is either a hydrate thereof or a pharmaceutically acceptable salt thereof. (Item 12) The composition according to item 11, wherein the compound of formula (IIIa), its hydrate, or a pharmaceutically acceptable salt thereof is present in an amount between about 0.001 ± 0.0002 wt% and about 2.0 ± 0.4 wt%. (Item 13) The compound of formula (IIIa), its hydrate, or a pharmaceutically acceptable salt thereof, in an amount between approximately 0.001±0.0002 wt% and approximately 2.0±0.4 wt%, or in an amount between approximately 0.001±0.0002 wt% and approximately 1.5±0.3 wt%, or in an amount between approximately 0.001±0.0002 wt% and approximately 1±0.2 wt%, or approximately 0.001±0.00 The composition according to item 11 or 12, present in an amount between 0.02 wt% and about 0.7 ± 0.14 wt%, or in an amount between about 0.001 ± 0.0002 wt% and about 0.5 ± 0.1 wt%, or in an amount between about 0.001 ± 0.0002 wt% and about 0.3 ± 0.06 wt%, or in an amount between about 0.001 ± 0.0002 wt% and about 0.2 ± 0.04 wt%. (Item 14) A composition according to any one of items 1 to 13, comprising the compound of formula (Ia) in an amount between approximately 97 wt% and approximately 100 wt%, or between approximately 98 wt% and approximately 100 wt%, or between approximately 99 wt% and approximately 100 wt%, on an anhydrous basis. (Item 15) The compound according to formula (III) is the compound according to formula (IV). [ka] The composition described in any one of items 1 to 9, or a stereoisomer thereof, hydrate, or pharmaceutically acceptable salt thereof. (Item 16) The compound of formula (IV), or its stereoisomer, hydrate, or pharmaceutically acceptable salt, in amounts between approximately 0.001±0.0002 wt% and approximately 2±0.4 wt%, or between approximately 0.001±0.0002 wt% and approximately 1.5±0.3 wt%, or between approximately 0.001±0.0002 wt% and approximately 1±0.2 wt%, or approximately 0.001±0 The composition according to item 15, present in an amount between 0.0002 wt% and about 0.7 ± 0.14 wt%, or in an amount between about 0.001 ± 0.0002 wt% and about 0.5 ± 0.1 wt%, or in an amount between about 0.001 ± 0.0002 wt% and about 0.3 ± 0.06 wt%, or in an amount between about 0.001 ± 0.0002 wt% and about 0.2 ± 0.04 wt%. (Item 17) The compound of formula (III) is the compound of formula (V). [ka] The composition described in any one of items 1-9 and 15-16, or a stereoisomer thereof, hydrate, or pharmaceutically acceptable salt thereof. (Item 18) The compound of formula (V), or its stereoisomer, hydrate, or pharmaceutically acceptable salt, is present in amounts between approximately 0.001±0.0002 wt% and approximately 2±0.4 wt%, or between approximately 0.001±0.0002 wt% and approximately 1.5±0.3 wt%, or between approximately 0.001±0.0002 wt% and approximately 1±0.2 wt%, or approximately 0.001±0 The composition described in item 17, present in an amount between 0.0002 wt% and about 0.7 ± 0.14 wt%, or in an amount between about 0.001 ± 0.0002 wt% and about 0.5 ± 0.1 wt%, or in an amount between about 0.001 ± 0.0002 wt% and about 0.3 ± 0.06 wt%, or in an amount between about 0.001 ± 0.0002 wt% and about 0.2 ± 0.04 wt%. (Item 19) The compound of formula (III) is the compound of formula (VI). [ka] The composition described in any one of items 1 to 9, or a stereoisomer thereof, hydrate, or pharmaceutically acceptable salt thereof. (Item 20) The compound of formula (VI), or its stereoisomer, hydrate, or pharmaceutically acceptable salt, is present in amounts between approximately 0.001±0.0002 wt% and approximately 2±0.4 wt%, or between approximately 0.001±0.0002 wt% and approximately 1.5±0.3 wt%, or between approximately 0.001±0.0002 wt% and approximately 1±0.2 wt%, or approximately 0.001±0 The composition according to item 19, present in an amount between 0.0002 wt% and about 0.7 ± 0.14 wt%, or in an amount between about 0.001 ± 0.0002 wt% and about 0.5 ± 0.1 wt%, or in an amount between about 0.001 ± 0.0002 wt% and about 0.3 ± 0.06 wt%, or in an amount between about 0.001 ± 0.0002 wt% and about 0.2 ± 0.04 wt%. (Item 21) The compound of formula (III) is the compound of formula (VII) [ka] The composition described in any one of items 1-9, 19, and 20, which is a stereoisomer thereof, a hydrate, or a pharmaceutically acceptable salt thereof. (Item 22) The compound of formula (VII), or its stereoisomer, hydrate, or pharmaceutically acceptable salt, is present in amounts between approximately 0.001±0.0002 wt% and approximately 2±0.4 wt%, or between approximately 0.001±0.0002 wt% and approximately 1.5±0.3 wt%, or between approximately 0.001±0.0002 wt% and approximately 1±0.2 wt%, or approximately 0.001± The composition according to item 21, present in an amount between 0.0002 wt% and about 0.7 ± 0.14 wt%, or in an amount between about 0.001 ± 0.0002 wt% and about 0.5 ± 0.1 wt%, or in an amount between about 0.001 ± 0.0002 wt% and about 0.3 ± 0.06 wt%, or in an amount between about 0.001 ± 0.0002 wt% and about 0.2 ± 0.04 wt%. (Item 23) The compound of formula (III) is the compound of formula (VIII) [ka] The composition according to any one of items 1-9 and 19-22, or a stereoisomer thereof, hydrate, or pharmaceutically acceptable salt thereof. (Item 24) The compound of formula (VIII), or its stereoisomer, hydrate, or pharmaceutically acceptable salt, is present in amounts between approximately 0.001±0.0002 wt% and approximately 2±0.4 wt%, or between approximately 0.001±0.0002 wt% and approximately 1.5±0.3 wt%, or between approximately 0.001±0.0002 wt% and approximately 1±0.2 wt%, or approximately 0.001± The composition according to item 23, present in an amount between 0.0002 wt% and about 0.7 ± 0.14 wt%, or in an amount between about 0.001 ± 0.0002 wt% and about 0.5 ± 0.1 wt%, or in an amount between about 0.001 ± 0.0002 wt% and about 0.3 ± 0.06 wt%, or in an amount between about 0.001 ± 0.0002 wt% and about 0.2 ± 0.04 wt%. (Item 25) Compound of formula (Ia) [ka] A composition comprising, a) Coupling H-MePro-OH with Z-Gly-OH in the presence of an activator, a silylation agent, and a solvent, or b) Coupling Z-Gly-OH with Suc-OH in the absence or presence of an activator and solvent, and in the absence or presence of a silylating agent, and then coupling the resulting Z-Gly-OSu with H-MePro-OH, c) Coupling the obtained Z-Gly-MePro-OH with H-Glu-OH in the presence of an activator, a silylation agent, and a solvent, d) To obtain Z-Gly-MePro-Glu-OH, e) Deprotecting Z-Gly-MePro-Glu-OH to obtain a composition comprising the compound of formula (Ia), and the composition obtained by this method. (Item 26) The composition according to item 25, wherein the obtained composition is dissolved or suspended in a solvent. (Item 27) The composition is a compound of formula (II). [ka] or further comprising its stereoisomer, hydrate, or pharmaceutically acceptable salt, and optionally a compound of formula (III). [ka] or comprising its stereoisomers, hydrates, solvates, or pharmaceutically acceptable salts, wherein R1, R2, R3, and R4 are independently hydrogen and C 1-4 Selected from the group consisting of alkyl groups, wherein at least one of R1, R2, R3, and R4 is C 1-4 A composition according to item 25 or 26, which is alkyl. (Item 28) The composition according to item 26 or 27, wherein the compound of formula (II), or its stereoisomers, hydrates, or pharmaceutically acceptable salts, is absent or present in an amount between approximately 0.001 ± 0.0002 wt% and approximately 2 ± 0.4 wt%, and the compound of formula (III), or its stereoisomers, hydrates, or pharmaceutically acceptable salts, is absent or present in an amount between approximately 0.001 ± 0.0002 wt% and approximately 2 ± 0.4 wt%, provided that at least one of the compounds of formula (II) or formula (III), or their stereoisomers, hydrates, or pharmaceutically acceptable salts, is present. (Item 29) A composition according to any one of items 26 to 28, wherein the compound of formula (II), or its stereoisomer, hydrate, or pharmaceutically acceptable salt is present in an amount between about 0.001 ± 0.0002 wt% and about 0.3 ± 0.06 wt%. (Item 30) A composition according to any one of items 26 to 29, wherein the compound of formula (II), or its stereoisomer, hydrate, or pharmaceutically acceptable salt is present in an amount between about 0.001 ± 0.0002 wt% and about 0.1 ± 0.02 wt%. (Item 31) The compound of formula (II), or its stereoisomer, hydrate, or pharmaceutically acceptable salt, is present in amounts between approximately 0.001±0.0002 wt% and approximately 2±0.4 wt%, or between approximately 0.001±0.0002 wt% and approximately 1.5±0.3 wt%, or between approximately 0.001±0.0002 wt% and approximately 1±0.2 wt%, or approximately 0.001±0.0 The composition according to item 26 or 27, present in an amount between 0.002 wt% and about 0.7 ± 0.14 wt%, or in an amount between about 0.001 ± 0.0002 wt% and about 0.5 ± 0.1 wt%, or in an amount between about 0.001 ± 0.0002 wt% and about 0.3 ± 0.06 wt%, or in an amount between about 0.001 ± 0.0002 wt% and about 0.2 ± 0.04 wt%. (Item 32) The compound according to formula (II) is the compound according to formula (IIa). [ka] The composition according to any one of items 26 to 31, which is either a hydrate or a pharmaceutically acceptable salt thereof. (Item 33) A composition according to any one of items 26 to 32, wherein the compound of formula (II), or its stereoisomer, hydrate, or pharmaceutically acceptable salt thereof, is not present. (Item 34) A composition according to any one of items 26 to 33, wherein the compound of formula (III), or its stereoisomer, hydrate, or pharmaceutically acceptable salt is present in an amount between about 0.001 ± 0.0002 wt% and about 2.0 ± 0.4 wt%. (Item 35) The composition according to any one of items 25 to 34, wherein the silylating agent does not contain a cyano group. (Item 36) The composition according to item 35, wherein the silylation agent is selected from the group consisting of N,O-bis(trimethylsilyl)acetamide, N,O-bis(trimethylsilyl)trifluoroacetamide, hexamethyldisilazane, N-methyl-N-(trimethylsilyl)acetamide, N-methyl-N-(trimethylsilyl)trifluoroacetamide, N-(trimethylsilyl)acetamide, N-(trimethylsilyl)diethylamine, N-(trimethylsilyl)dimethylamine, 1-(trimethylsilyl)imidazole, and 3-(trimethylsilyl)2-oxazolidone. (Item 37) The composition according to any one of items 25 to 36, wherein the solvent in step a) is a polar organic solvent or a polar aprotic organic solvent. (Item 38) The composition according to any one of items 25 to 37, wherein the solvent in step b) is a polar organic solvent or a polar aprotic organic solvent. (Item 39) The composition according to any one of items 25 to 38, wherein the solvent in step c) is a polar organic solvent or a polar aprotic organic solvent. (Item 40) The composition according to any one of items 25 to 39, wherein the solvent is selected from the group consisting of alkyl acetates, chlorinated hydrocarbons, alkyl cyanides, and amide solvents, or mixtures thereof. (Item 41) The composition according to any one of items 25 to 40, wherein the activator is selected from the group consisting of carbodiimide, acyl halide, phosphonium salt, uronium salt, and guanidinium salt. (Item 42) A composition according to any one of items 25 to 41, wherein deprotection is achieved by hydrogenation. (Item 43) The composition according to item 42, wherein the hydrogenation is carried out in the presence of a Pd / C catalyst. (Item 44) The composition according to item 42, wherein the hydrogenation is carried out in the presence of a Pd / Si catalyst. (Item 45) The composition according to any one of items 42 to 44, wherein the hydrogenation is carried out at a temperature of about 10 to 40°C, or at a temperature of about 20 to 30°C, or at a temperature of about 25°C. (Item 46) The composition according to any one of items 42 to 45, wherein the hydrogenation is carried out at a pressure of about 0 to about 6 bar. (Item 47) The composition according to any one of items 42 to 46, wherein the hydrogenation is carried out using at least one solvent selected from the group consisting of water, ethyl acetate, isopropyl acetate, methanol, ethanol, isopropanol, or mixtures thereof. (Item 48) The following steps a) Coupling H-MePro-OH with Z-Gly-OH in the presence of an activator, a silylation agent, and a solvent, or b) Coupling Z-Gly-OH with Suc-OH in the absence or presence of an activator and solvent, and in the absence or presence of a silylating agent, and then coupling the resulting Z-Gly-OSu with H-MePro-OH, c) Coupling the obtained Z-Gly-MePro-OH with H-Glu-OH in the presence of an activator, a silylation agent, and a solvent, d) To obtain Z-Gly-MePro-Glu-OH, e) A method for producing a composition containing trofinetide, comprising the steps of: deprotecting Z-Gly-MePro-Glu-OH to produce trofinetide, wherein the trofinetide is dissolved or suspended in a solvent. (Item 49) The method according to item 48, wherein the silylating agent does not contain a cyano group. (Item 50) The method according to item 48 or 49, wherein the silylation agent is selected from the group consisting of N,O-bis(trimethylsilyl)acetamide, N,O-bis(trimethylsilyl)trifluoroacetamide, hexamethyldisilazane, N-methyl-N-(trimethylsilyl)acetamide (TMA), N-methyl-N-(trimethylsilyl)trifluoroacetamide, N-(trimethylsilyl)acetamide, N-(trimethylsilyl)diethylamine, N-(trimethylsilyl)dimethylamine, 1-(trimethylsilyl)imidazole, and 3-(trimethylsilyl)2-oxazolidone. (Item 51) The method according to any one of items 48 to 50, wherein the solvent in step a) is a polar organic solvent or a polar aprotic organic solvent. (Item 52) The method according to any one of items 48 to 51, wherein the solvent in step b) is a polar organic solvent or a polar aprotic organic solvent. (Item 53) The method according to any one of items 48 to 52, wherein the solvent in step c) is a polar organic solvent or a polar aprotic organic solvent. (Item 54) The method according to any one of items 48 to 53, wherein the solvent is selected from the group consisting of alkyl acetates, chlorinated hydrocarbons, alkyl cyanides, and amide solvents, or mixtures thereof. (Item 55) The method according to any one of items 48 to 54, wherein the activator is selected from the group consisting of carbodiimide, acyl halide, phosphonium salt, uronium salt, and guanidinium salt. (Item 56) The method according to any one of items 48 to 55, wherein deprotection is achieved by hydrogenation. (Item 57) The method according to item 56, wherein the hydrogenation is carried out in the presence of a Pd / C catalyst. (Item 58) The method according to item 56, wherein the hydrogenation is carried out in the presence of a Pd / Si catalyst. (Item 59) The method according to any one of items 56 to 58, wherein the hydrogenation is carried out at a temperature of about 10 to 40°C, or at a temperature of about 20 to 30°C, or at a temperature of about 25°C. (Item 60) The method according to any one of items 56 to 59, wherein the hydrogenation is carried out at a pressure of about 0 to about 6 bar. (Item 61) The method according to any one of items 56 to 60, wherein the hydrogenation is carried out using at least one solvent selected from the group consisting of water, ethyl acetate, isopropyl acetate, methanol, ethanol, isopropanol, or mixtures thereof. (Item 62) A product comprising any one of the compositions described in item 1 to 47. (Item 63) The aforementioned product is a kit, as described in item 62. (Item 64) Compound of formula (Ia) [ka] The hydrate or pharmaceutically acceptable salt thereof, and the compound of formula (IIa). [ka] A kit comprising a dosage form containing a hydrate or a pharmaceutically acceptable salt thereof, and instructions for use for administration to a subject requiring it. (Item 65) The kit described in item 64 further comprises one or more compounds selected from the group consisting of formulas (III), (IIIa), (IV), (V), (VI), (VII), (VIII), and (IX). (Item 66) A composition according to any one of items 1 to 47, comprising, on an anhydrous basis, at least 90 wt%, for example, 91 wt%, 92 wt%, 93 wt%, 94 wt%, 95 wt%, 96 wt%, and 97 wt%, of the compound of formula (I) or its stereoisomer, hydrate, or pharmaceutically acceptable salt. (Item 67) A composition according to any one of items 2 to 47, comprising, on an anhydrous basis, at least 90 wt%, for example, 91 wt%, 92 wt%, 93 wt%, 94 wt%, 95 wt%, 96 wt%, and 97 wt%, of the compound of formula (Ia) or its stereoisomer, hydrate, or pharmaceutically acceptable salt. (Item 68) Compound of formula (I) [ka] or its stereoisomers, hydrates, or pharmaceutically acceptable salts, and compounds of formula (II). [ka] or its stereoisomers, hydrates, or pharmaceutically acceptable salts, and / or compounds of formula (III). [ka] or its stereoisomers, hydrates, or pharmaceutically acceptable salts, wherein R1, R2, R3, and R4 are independently hydrogen and C 1-4 Selected from the group consisting of alkyl groups, wherein at least one of R1, R2, R3, and R4 is C 1-4 A composition comprising an alkyl compound, wherein the composition, on an anhydrous basis, comprises at least 90 wt%, for example, 91 wt%, 92 wt%, 93 wt%, 94 wt%, 95 wt%, 96 wt%, and 97 wt%, of the compound of formula (I). (Item 69) Compound of formula (Ia) [ka] or its hydrate, or a pharmaceutically acceptable salt thereof, and formula (II) [ka] or its stereoisomers, hydrates, or pharmaceutically acceptable salts, and / or compounds of formula (III). [ka] A composition comprising either a stereoisomer thereof, a hydrate, or a pharmaceutically acceptable salt thereof, wherein R1, R2, R3, and R4 are independently hydrogen and C 1-4 Selected from the group consisting of alkyl groups, wherein at least one of R1, R2, R3, and R4 is C 1-4 The composition is alkyl, and on an anhydrous basis, comprises at least 90 wt%, for example 91 wt%, 92 wt%, 93 wt%, 94 wt%, 95 wt%, 96 wt%, and 97 wt% of the compound of formula (Ia). (Item 70) Compound of formula (Ia) [ka] or its hydrate, or a pharmaceutically acceptable salt thereof, and the compound of formula (IIa). [ka] A product comprising a kit comprising a dosage form comprising the hydrate or pharmaceutically acceptable salt thereof, and having instructions for use thereof, wherein the product contains a specified amount of the compound of formula (Ia) in the product between 95 wt% and 105 wt%, for example, 96 wt%, 97 wt%, 98 wt%, 99 wt%, 100 wt%, 101 wt%, 102 wt%, 103 wt%, or 104 wt%. (Item 71) Compound of formula (Ia) [ka] or its hydrate, or a pharmaceutically acceptable salt thereof, and the compound of formula (IIa). [ka] A product comprising a kit comprising a dosage form with instructions for use, further comprising one or more compounds selected from the group consisting of formulas (III), (IIIa), (IV), (V), (VI), (VII), (VIII), and (IX), wherein the composition comprises between 95 wt% and 105 wt% of a specified amount of the compound of formula (Ia) in the product, for example, 96 wt%, 97 wt%, 98 wt%, 99 wt%, 100 wt%, 101 wt%, 102 wt%, 103 wt%, or 104 wt%.

Claims

1. Compound of formula (I) 【Chemistry 101】 or its stereoisomers, or pharmaceutically acceptable salts, and compounds of formula (II) between approximately 0.001 wt% and approximately 2 wt%. 【Chemical Engineering 102】 A composition containing the following:

2. The compound according to formula (I) is the compound according to formula (Ia). 【Chemistry 103】 The composition according to claim 1, or a pharmaceutically acceptable salt thereof.

3. The compound according to formula (II) is the compound according to formula (IIa). 【Chemical 104】 The composition according to claim 1, or a pharmaceutically acceptable salt thereof.

4. The composition is a) Coupling (2S)-2-methylpyrrolidine-2-carboxylic acid with benzyloxycarbonylglycine in the presence of an activator, a silylation agent, and a solvent, or b) Coupling benzyloxycarbonyl-glycine with N-hydroxysuccinimide in the absence or presence of an activator and solvent, and in the absence or presence of a silylating agent, and then coupling the resulting benzyloxycarbonyl-glycine N-succinimidyl ester with (2S)-2-methylpyrrolidine-2-carboxylic acid, c) Coupling the obtained ((S-1-(((benzyloxy)carbonyl)glycyl)-2-methylpyrrolidine-2-carboxylic acid with (2S)-2-aminopentanediic acid in the presence of an activator, a silylater, and a solvent, d) To obtain ((S)-1-(((benzyloxycarbonyl)glycyl)-2-methylpyrrolidine-2-carbonyl)-L-glutamic acid, e) Deprotecting ((S)-1-(((benzyloxy)carbonyl)glycyl)-2-methylpyrrolidine-2-carbonyl)-L-glutamic acid to obtain a composition containing the compound of formula (Ia), The composition according to claim 2, obtained by the means described herein.

5. The compound according to formula (II) is the compound according to formula (IIa). 【Chemistry 105】 The composition according to claim 4, or a pharmaceutically acceptable salt thereof.

6. The composition according to claim 4, wherein deprotection is achieved by hydrogenation.

7. The composition according to claim 6, wherein the hydrogenation is carried out in the presence of a Pd / C catalyst.

8. The composition according to claim 6, wherein the hydrogenation is carried out in the presence of a Pd / Si catalyst.

9. The composition according to claim 6, wherein the hydrogenation is carried out using at least one solvent selected from the group consisting of water, ethyl acetate, isopropyl acetate, methanol, ethanol, and isopropanol, or mixtures thereof.

10. The composition according to claim 1, wherein the compound of formula (II), its stereoisomer, or a pharmaceutically acceptable salt is present in an amount between about 0.001 wt% and about 0.2 wt%.

11. Compound of formula (Ia) 【Chemistry 106】 or a pharmaceutically acceptable salt thereof, and compounds of formula (IIa) between about 0.001 wt% and about 0.2 wt%. 【Chemistry 107】 A composition comprising a pharmaceutically acceptable salt thereof.

12. The composition according to claim 1, wherein the compound of formula (II) or its stereoisomer, or a pharmaceutically acceptable salt thereof, is present in an amount between about 0.001 wt% and about 1 wt%.

13. The composition according to claim 1, wherein the compound of formula (II) or its stereoisomer, or a pharmaceutically acceptable salt thereof, is present in an amount between about 0.001 wt% and about 0.5 wt%.

14. The composition according to claim 1, wherein the compound of formula (II) or its stereoisomer, or a pharmaceutically acceptable salt thereof, is present in an amount between about 0.001 wt% and about 0.3 wt%.

15. The composition according to claim 1, wherein the compound of formula (II) or its stereoisomer, or a pharmaceutically acceptable salt thereof, is present in an amount between about 0.001 wt% and about 0.1 wt%.

16. The composition according to claim 5, wherein the compound of formula (IIa) or its stereoisomer, or a pharmaceutically acceptable salt thereof, is present in an amount between about 0.001 wt% and about 1 wt%.

17. The composition according to claim 5, wherein the compound of formula (IIa) or its stereoisomer, or a pharmaceutically acceptable salt thereof, is present in an amount between about 0.001 wt% and about 0.5 wt%.

18. The composition according to claim 5, wherein the compound of formula (IIa) or its stereoisomer, or a pharmaceutically acceptable salt thereof, is present in an amount between about 0.001 wt% and about 0.3 wt%.

19. The composition according to claim 5, wherein the compound of formula (IIa) or its stereoisomer, or a pharmaceutically acceptable salt thereof, is present in an amount between about 0.001 wt% and about 0.1 wt%.

20. The composition according to claim 2, comprising, on an anhydrous basis, a compound of formula (Ia) in an amount between about 98 wt% and about 100 wt%.

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