EA049473B1 - COMPOSITIONS OF TROPHINETIDE - Google Patents

Description

Field of invention

The present invention relates to compositions and methods of preparation comprising trofinetide (glycyl-E-2-methylprolyl-E-glutamic acid, or G-2-MePE). The compositions are prepared using new preparation methods and contain trofinetide and other products of the synthetic methods.

State of the art

Trofinetide is a synthetic compound that has a similar core structure to glycyl-prolyl-glutamic acid (or GPE). Trofinetide has been found to be useful in the treatment of neurodegenerative conditions and has recently been found to be effective in the treatment of autism spectrum disorders and neurodevelopmental disorders.

The essence of the invention

The inventors identified a new problem in this area, namely, that current methods for producing compositions containing trofinetide are difficult to scale up to commercial quantities.

To solve this problem, the inventors have identified new compositions and methods of preparation in which the compound, or a salt or stereoisomer or hydrate thereof, is prepared using silylation technology. Using this new strategy provides new compositions and substantially higher amounts of trofinetide, and results in compositions, kits, and products containing trofinetide that are commercially useful.

The present invention relates to a composition comprising a compound of formula (I)

or its stereoisomer, hydrate or pharmaceutically acceptable salt, and a compound of formula (II): O g——·\

^it (II), or its stereoisomer, hydrate or pharmaceutically acceptable salt, and/or a compound of formula (III):

or a stereoisomer, hydrate or pharmaceutically acceptable salt thereof, wherein R _b R ₂ , R3 and R ₄ are independently selected from the group consisting of hydrogen and C1.4 alkyl, provided that at least one of Ri, R ₂ , R3 and R ₄ is Ci. ₄ alkyl.

The present invention also relates to a compound of formula (1a) (trofinetide)

- 1 049473

or its hydrate or pharmaceutically acceptable salt.

The present invention also relates to a composition containing trofinetide, said composition being obtained by:

a) coupling reactions between H-MePro-OH and Z-Gly-OH in the presence of an activating reagent, a silylating agent and a solvent, or

b) coupling reactions between Z-Gly-OH and Suc-OH, and then coupling reactions between the resulting ZGly-OSu and H-MePrO-OH in the absence or in the presence of an activating reagent, a solvent, and in the absence or in the presence of a silylating agent;

c) coupling reactions between the obtained Z-Gly-MePro-OH and H-Glu-OH in the presence of an activating reagent, a silylating agent and a solvent;

d) obtaining Z-Gly-MePro-Glu-OH; and

e) removing the protecting group from Z-Gly-MePro-Glu-OH to obtain a composition containing trofinetide.

The present invention also relates to a method for producing a composition containing trofinetide, comprising the steps of:

a) coupling reactions between H-MePro-OH and Z-Gly-OH in the presence of an activating reagent, a silylating agent and a solvent, or

b) coupling reactions between Z-Gly-OH and Suc-OH, and then coupling reactions between the resulting ZGly-OSu and H-MePrO-OH in the absence or in the presence of an activating reagent, a solvent, and in the absence or in the presence of a silylating agent;

c) coupling reactions between the obtained Z-Gly-MePro-OH and H-Glu-OH in the presence of an activating reagent, a silylating agent and a solvent;

d) obtaining Z-Gly-MePro-Glu-OH; and

e) removing the protecting group from Z-Gly-MePro-Glu-OH to obtain a composition containing trofinetide.

The present invention also relates to a kit comprising a dosage form comprising a trofinetide compound and a compound of formula (Pa)

o (Pa) (Z-Gly-MePro-Glu-OH), or its hydrate or pharmaceutically acceptable salt, and instructions for use.

The present invention also relates to a kit comprising a dosage form comprising a compound of formula (1a) and a compound of formula (Ia), and one or more compounds selected from the group consisting of compounds of formula (III), formula (IIIa), formula (IV), formula (V), formula (VI), formula (VII), formula (VIII) and formula (IX).

Detailed description

The synthesis of peptides 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 persilylation agents selected from the group consisting of N,Obis(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.

Applicability

Trofinetide has been shown to be useful for the treatment of neurodegenerative conditions, neurodevelopmental disorders and autism spectrum disorders.

Definitions

The following definitions shall be understood as set out below, unless they are defined con

-2049473 specifically anywhere else in this description.

The terms Cbz and Z stand for benzyloxycarbonyl protecting group and are interchangeable and have the following structure:

The term trofinetide means glycyl-T-2-methylprolyl-T-glutamic acid of formula (1a) or G-2-MePE, or H-Gly-MePro-Glu-OH, or Gly-MePro-Glu-OH, where the chiral centers have the S,S configuration

(1a).

The IUCN name for trofinetide is (28)-2-[[(28)-1-(2-aminoacetyl)-2-methylpyrrolidine-2-carbonyl]amino]pentanedioic acid.

The term approximately means the mean value of the variable is ±20% of the mean value of the variable.

The term eq. means equivalent. Unless otherwise specifically stated, all equivalents (eq.) are given relative to the limiting reagent on a molar basis.

The term vol. means volume and volumes.

The term DIPEA stands for N,N-diisopropylethylamine, or Hunig's base.

The term DMAC stands for N,N-dimethylacetamide.

The term NMT means nothing more than.

The term TMA stands for N-methyl-N-(trimethylsilyl)acetamide.

The term BSA stands for N,O-bis(trimethylsilyl)acetamide.

The term Oxyma Pure stands for ethyl 2-cyano-2-(hydroxyimino)acetate, or ethyl (hydroxyimino) cyanoacetate.

The term IPE stands for isopropyl ether or diisopropyl ether.

The term ACN stands for acetonitrile.

The term NLT means not less than.

The term MTBE stands for 2-methoxy-2-methylpropane, or methyl tert-butyl ether.

The term iPrOH stands for propan-2-ol, or isopropyl alcohol, or isopropanol.

The term iPrOAc stands for isopropyl acetate.

The term UHPLC stands for ultra-high performance liquid chromatography.

The term Piv-Cl stands for pivaloyl chloride.

The term EtOAc or AcOEt stands for ethyl acetate.

The term HPLC stands for high performance liquid chromatography.

The term MeOH stands for methyl alcohol or methanol.

The term Pd/C stands for palladium on carbon catalyst.

The term Pd/Si refers to palladium catalyst on silicon dioxide.

The term RT refers to the ambient temperature, typically approximately 25°C.

The term LC stands for liquid chromatography.

The term RRT stands for relative retention time.

The term TFA stands for trifluoroacetic acid.

The term H-MePrO-OH means (28)-2-methylpyrrolidine-2-carboxylic acid, either in the form of the base or in the form of the hydrochloric acid salt.

The term MePro.NSG stands for (28)-2-methylpyrrolidine-2-carboxylic acid hydrochloride.

The term Z-Gly-OH stands for benzyloxycarbonyl-glycine, or Cbz-glycine.

The term H-Glu-OH stands for (2S)-2-aminopentanedioic acid, or L-glutamic acid.

The term EDC.HC1 means 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride or N-(3-dimethylaminopropyl)-X'-ethylcarbodiimide hydrochloride.

The term Z-Gly-MePro-OH means ((8)-1-(((benzyloxy)carbonyl)glycyl)-2-methylpyrrolidine-2-carboxylic acid) or X-benzyloxycarbonyl-glycyl-E-2-methylproline.

The term 'Z-Gly-MePro-Glu-OH' (methylpyrrolidine-2-carbonyl)-E-glutamic acid means ((8)-1-(((benzyloxy)carbonyl)glycyl)-2 acid, or 18G-benzyloxycarbonyl-glycyl-E-2

-3 049473 methylprolyl-E-glutamic acid.

The term Et-Gly-MePro-Glu-OH means ((8)-1-(ethylglycyl)-2-methylpyrrolidine-2-carbonyl)-L-glutamic acid, or N-ethylglycyl-E-2-methylprolyl-E-glutamic acid.

The term Me-Gly-MePro-Glu-OH means ((p)-1-(methylglycyl)-2-methylpyrrolidine-2-carbonyl)-E-glutamic acid, or methyl-glycyl-E-2-methylprolyl-E-glutamic acid.

The term Me ₂ -Gly-MePro-Glu-OH means (N)-1-(dimethylglycyl)-2-methylpyrrolidine-2-carbonyl)-E-glutamic acid, or N,N-dimethylglycyl-E-2-methylprolyl-E-glutamic acid.

The term Gly-MePro-Glu(OiPr)-OH means 5-isopropyl ester of glycyl-E-2-methylprolyl-E-glutamic acid, or γ-isopropyl ester of glycyl-E-2-methylprolyl-E-glutamic acid.

The term Gly-MePro-Glu-OiPr means 1-isopropyl ester of glycyl-E-2-methylprolyl-E-glutamic acid, or α-isopropyl ester of glycyl-E-2-methylprolyl-E-glutamic acid.

The term Gly-MePro-Glu(OiPr)-OiPr means glycyl-E-2-methylprolyl-E-glutamic acid 1,5-diisopropyl ester.

The term Gly-MePro-Glu(OMe)-OH means glycyl-E-2-methylprolyl-E-glutamic acid 5-methyl ester, or glycyl-E-2-methylprolyl-E-glutamic acid γ-methyl ester.

The term Gly-MePro-Glu-OMe means glycyl-E-2-methylprolyl-E-glutamic acid 1-methyl ester, or glycyl-E-2-methylprolyl-E-glutamic acid α-methyl ester.

The term Gly-MePro-Glu(OMe)-OMe stands for glycyl-E-2-methylprolyl-E-glutamic acid 1,5-dimethyl ester.

The term Gly-MePro-Glu(OEt)-OH means 5-ethyl ester of glycyl-E-2-methylprolyl-E-glutamic acid, or γ-ethyl ester of glycyl-E-2-methylprolyl-E-glutamic acid.

The term Gly-MePro-Glu-OEt means 1-ethyl ester of glycyl-E-2-methylprolyl-E-glutamic acid, or α-ethyl ester of glycyl-E-2-methylprolyl-E-glutamic acid.

The term Gly-MePro-Glu(OEt)-OEt means glycyl-E-2-methylprolyl-E-glutamic acid 1,5-diethyl ester.

The term Suc-OH stands for N-hydroxysuccinimide.

The term Z-Gly-OSu stands for benzyloxycarbonylglycine N-succinimidyl ester.

The term DCC stands for N,N'-dicyclohexylcarbodiimide.

The term DCU stands for 1,3-dicyclohexylurea.

The term TEA stands for triethylamine.

The term CH ₂ Cl ₂ stands for DCM, or dichloromethane.

The term NaHCO ₃ stands for sodium bicarbonate.

The term iButOH stands for butan-2-ol, or isobutyl alcohol or isobutanol.

The term DMAPA stands for dimethylaminopropylamine.

The term KHSO ₄ stands for potassium bisulfate.

The term H2O means water.

The term NaCl means sodium chloride, or salt.

The term DMA stands for N,N-dimethylacetamide.

The term DMF stands for N,N-dimethylformamide.

The term HCl stands for hydrochloric acid.

The term HOBt stands for 1-hydroxybenzotriazole.

The term HOAt stands for 1-hydroxyazabenzotriazole.

The term TBTU stands for 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate.

The term NMM stands for N-methylmorpholine.

The term ICH stands for International Commission on Harmonization.

The term NMR stands for nuclear magnetic resonance.

The term UV stands for ultraviolet radiation.

The term MS stands for mass spectrometry.

The term g stands for gram.

The term µl means microliter.

The terms activating reagent and carboxyl group activating reagent mean a reagent that promotes the linkage of two amino acids and/or peptides by forming an amide bond. Examples of activating agents and carboxyl group activating agents include carbodiimides, acyl halides, phosphonium salts and uronium or guanidinium salts, acyl halides such as isobutyl chloroformate or pivaloyl chloride, or a carbodiimide such as EDC.HCl or DCC.

The term containing means includes, but is not limited to.

The term consisting of means including only the specified material.

The term "consisting essentially of" means including the specified material and its equivalents.

The terms % w/w and wt% are used interchangeably in this description.

The term specified amount means the amount of a compound expressed in units of mass.

- 4 049 473 sa/volume or mass/mass, which is believed to be present in the product. The actual amount of the compound may exceed 100 wt.% of the specified amount if it contains an excess to compensate for loss of the compound during storage, for example through destruction or degradation of the compound.

The term persilylation agent or silylating agent refers to a compound that adds silicon atoms to a peptide or amino acid.

The term persilylated means a compound in which the groups having an active hydrogen atom that can react with the silylating agent are sufficiently silylated to ensure that a uniform reaction environment is achieved.

The term functional groups to be silylated is intended to refer to specific groups having a hydrogen atom that reacts with the silylating agent, such as amino, hydroxyl, mercapto or carboxyl groups.

The term by-product(s) means compounds of formulae (II), (III), (IIIa), (IV), (V), (VI), (VII), (VIII) and (IX).

General description

The present invention relates to methods for preparing compounds of formula (I), compounds and compositions 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), and to products comprising compositions comprising one or more compounds of these formulas. The general approach described herein involves the synthesis of compounds using silylating agents. Based on the chirality of the starting materials, mixtures of compounds can be prepared, wherein the majority of the compounds have the chirality of the starting materials and some additional compounds having different chirality.

These compounds and compositions are useful for the treatment of various neurodegenerative disorders, autism spectrum disorders and neurodevelopmental disorders.

Compounds of the present invention

The following compounds are to be considered independent of each other, but they may be combined with any other compounds. They include compounds of formula (I) and compounds of formula (II), (Ia), (III), (IIIa), (IV), (V), (VI), (VII), (VIII) and (IX).

Compound of formula (I) h ₂ n

^it (I), or a hydrate or a pharmaceutically acceptable salt thereof, wherein the chiral centers have the configurations S, S, S, R, R, S or R, R and wherein the chiral center is designated by * in formula (I). The chiral centers of the compounds of formulas (Ia), (II), (Ia), (III), (IIIa), (IV), (V), (VI), (VII), (VIII) and (IX) are the same as for formula (I).

Compound of formula (1a)

ЗН ^υΗ (la) (trofinetide), or its hydrate or pharmaceutically acceptable salt, wherein the chiral centers have the S, S configurations.

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Compound of formula (II)

or a hydrate or pharmaceutically acceptable salt thereof, wherein the chiral centers have the configuration S, S, S, R, R, S or R, R.

Compound of formula (Pa)

™ (Pa) Z-Gly-MePro-Glu-OH, or a hydrate or pharmaceutically acceptable salt thereof, wherein the chiral centers have S, S configurations.

Compound of formula (III) o

o / Rj (III), or a hydrate or a pharmaceutically acceptable salt thereof, wherein the chiral centers have the configurations S, S, S, R, R, S or R, R, and where R|. R ₂ , R ₃ and R ₄ are independently selected from the group consisting of hydrogen and Ci-4 alkyl, provided that at least one of R _b R ₂ , R ₃ and R4 is C1.4 alkyl.

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

A compound of formula (III) containing Gly-MePro-Glu-Ome, which means 1-methyl ester of glycyl-E-2-methylprolyl-E-glutamic acid or α-methyl ester of glycyl-L-2-methylprolyl-L-glutamic acid.

A compound of formula (III) containing Gly-MePro-Glu(OMe)-Ome, which means 1,5-dimethyl ester of glycyl-L-2-methylprolyl-L-glutamic acid.

A compound of formula (III) containing Gly-MePro-Glu(OEt)-OH, which means 5-ethyl ester of glycyl-L-2-methylprolyl-L-glutamic acid, or γ-ethyl ester of glycyl-L-2-methylprolyl-L-glutamic acid.

A compound of formula (III) containing Gly-MePro-Glu-OEt, which means 1-ethyl ester of glycyl-E-2-methylprolyl-E-glutamic acid, or oc-ethyl ester of glycyl-E-2-methylprolyl-L-glutamic acid.

A compound of formula (III) containing Gly-MePro-Glu(OEt)-OEt, which means 1,5-diethyl ester of glycyl-E-2-methylprolyl-L-glutamic acid.

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Compound of formula (Ша)

he (Sh) Et-Gly-MePro-Glu-OH, or a hydrate or pharmaceutically acceptable salt thereof, wherein the chiral centers have the S, S configuration.

Compound of formula (IV)

(IV) (Me-Gly-MePro-Glu), or a hydrate or pharmaceutically acceptable salt thereof, wherein the chiral centers have the S, S, S, R, R, S or R, R configuration.

Compound of formula (V)

it (V) (Me ₂ -Gly-MePro-Glu), or a hydrate or pharmaceutically acceptable salt thereof, wherein the chiral centers have the S, S, S, R, R, S or R, R configurations.

Compound of formula (VI)

X (VI) (Gly-MePro-Glu(OiPr)-OH), or a hydrate or pharmaceutically acceptable salt thereof, wherein the chiral centers have the configuration S, S, S, R, R, S or R, R.

-7 049473

Compound of formula (VII)

(VII) (Gly-MePro-Glu-OiPr), or a hydrate or pharmaceutically acceptable salt thereof, wherein the chiral centers have the S, S, S, R, R, S or R, R configuration.

Compound of formula (VIII)

\ (VIII) (Gly-MePro-Glu-(OiPr) ₂ ), or a hydrate or pharmaceutically acceptable salt thereof, wherein the chiral centers have the S, S, S, R, R, S or R, R configuration.

Compound of formula (IX)

or a hydrate or pharmaceutically acceptable salt thereof, wherein the chiral centers have the configuration S, S, S, R, R, S or R, R.

Compounds of formulas (II), (Ia), (III), (IIIa), (IV), (V), (VI), (VII), (VIII) and (IX), whenever present, are by-products in the compositions described herein.

Compositions, preparation and products of the present invention

The products of the present invention relate to unisolated materials, isolated materials, chemical intermediates, formulations, dosage forms, kits, instructions, and the like for use that include the compositions of the present invention. The compositions and products of the present invention include the following:

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.

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, and/or a compound of formula (III), its stereoisomer, hydrate or pharmaceutically acceptable salt.

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, and/or a compound of formula (IX), its stereoisomer, hydrate or pharmaceutically acceptable salt.

A composition comprising a compound of formula (1a) (trofinetide), its hydrate or pharmaceutically acceptable salt, and at least a compound of formula (Ia), its hydrate or pharmaceutically acceptable salt.

A composition containing a compound of formula (1a) (trofinetide) and a compound of formula (Pa).

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A composition containing a compound of formula (1a) (trofinetide), its hydrate or pharmaceutically acceptable salt, and a compound of formula (Ia), its hydrate or pharmaceutically acceptable salt, and/or a compound of formula (IIIa), its hydrate or pharmaceutically acceptable salt.

A composition containing a compound of formula (1a) (trofinetide), its hydrate or pharmaceutically acceptable salt, and a compound of formula (IIa), its hydrate or pharmaceutically acceptable salt, and/or a compound of formula (III), its hydrate or pharmaceutically acceptable salt.

A composition containing a compound of formula (1a) (trofinetide) and a compound of formula (II) and/or a compound of formula (III).

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, and/or a compound of formula (IV), its stereoisomer, hydrate or pharmaceutically acceptable salt.

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, and/or a compound of formula (V), its stereoisomer, hydrate or pharmaceutically acceptable salt.

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, and/or a compound of formula (VI), its stereoisomer, hydrate or pharmaceutically acceptable salt.

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, and/or a compound of formula (VII), its stereoisomer, hydrate or pharmaceutically acceptable salt.

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, and/or a compound of formula (VHI), its stereoisomer, hydrate or pharmaceutically acceptable salt.

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

A composition comprising a compound of formula (1a), its hydrate or pharmaceutically acceptable salt, and a compound of formula (IIIa).

A kit containing a dosage form containing a compound of formula (1a) (trofinetide) and a compound of formula (Pa), and instructions for use.

The kit may further comprise one or more compounds selected from the group consisting of compounds of formula (III), formula (IIIa), formula (IV), formula (V), formula (VI), formula (VII), formula (VIII) and formula (IX).

Implementation options

A composition according to any one of the following embodiments comprising a compound of formula (I)

o or its stereoisomer, hydrate or pharmaceutically acceptable salt, and a compound of formula (II):

-9049473

or its stereoisomer, hydrate or pharmaceutically acceptable salt, and/or a compound of formula (III):

Ri

(III), or a stereoisomer, hydrate or pharmaceutically acceptable salt thereof, wherein R _b R ₂ , R3 and R ₄ are independently selected from the group consisting of hydrogen and Ci. ₄ alkyl, provided that at least one of Ri, R ₂ , R ₃ and R ₄ is Ci. ₄ alkyl.

A composition according to any of the preceding or following embodiments, wherein the compound according to formula (I) is a compound of formula (1a).

(la) (trofinetide), or a hydrate or pharmaceutically acceptable salt thereof.

A composition according to any of the preceding or following embodiments, wherein the compound of formula (II) or a stereoisomer, hydrate, or pharmaceutically acceptable salt thereof is absent or present in an amount of from about 0.001±0.0002 wt.% to about 2±0.4 wt.%; and the compound of formula (III) or a stereoisomer, hydrate, or pharmaceutically acceptable salt thereof is absent or present in an amount of from about 0.001±0.0002 wt.% to about 2±0.4 wt.%, provided that at least one of the compounds of formula (II) or (III), or a stereoisomer, hydrate, or pharmaceutically acceptable salt thereof, is present.

A composition according to any of the preceding or following embodiments, wherein the compound of formula (II) or a stereoisomer, hydrate, or pharmaceutically acceptable salt thereof is present in an amount of from about 0.001±0.0002 wt.% to about 0.3±0.06 wt.%.

A composition according to any of the preceding or following embodiments, wherein the compound of formula (II) or a stereoisomer, hydrate, or pharmaceutically acceptable salt thereof is present in an amount of from about 0.001±0.0002 wt.% to about 0.1±0.02 wt.%.

A composition according to any one of the preceding or following embodiments, wherein the compound of formula (II) or a stereoisomer, hydrate, or pharmaceutically acceptable salt thereof is present in an amount of from about 0.001±0.0002% by weight to about 2±0.4% by weight, or from about 0.001±0.0002% by weight to about 1.5±0.3% by weight, or from about 0.001±0.0002% by weight to about 1±0.2% by weight, or from about 0.001±0.0002% by weight to about 0.7±0.14% by weight, or from about 0.001±0.0002% by weight to about 0.5±0.1% by weight, or from about 0.001±0.0002% by weight to about 0.3±0.06% by weight, or from about 0.001±0.0002 wt.% to about 0.2±0.04 wt.%.

A composition according to any one of the preceding or following embodiments, wherein the compound of formula (II) is a compound of formula (IIa):

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he (Pa) (Z-Gly-MePro-Glu-OH), or its hydrate or pharmaceutically acceptable salt.

A composition according to any of the preceding or following embodiments, wherein the compound of formula (II) or a stereoisomer, hydrate or pharmaceutically acceptable salt thereof is absent.

A composition according to any of the preceding or following embodiments, wherein the compound of formula (III) or a stereoisomer, hydrate or pharmaceutically acceptable salt thereof is present in an amount of from about 0.001±0.0002 wt.% to about 2.0±0.4 wt.%.

A composition according to any one of the preceding or following embodiments, wherein said composition comprises a compound of formula (III), wherein the compound of formula (III) is a compound of formula (IX),

or its hydrate or pharmaceutically acceptable salt.

A composition according to any one of the preceding or following embodiments, comprising a compound of formula (III), wherein the compound of formula (III) is a compound of formula (IIIa):

he he (Sha) (Et-Gly-MePro-Glu-OH), or its hydrate or pharmaceutically acceptable salt.

A composition according to any of the preceding or following embodiments, wherein the compound of formula (IIIa) or a hydrate or pharmaceutically acceptable salt thereof is present in an amount of from about 0.001±0.0002 wt.% to about 2.0±0.4 wt.%.

A composition according to any one of the preceding or following embodiments, wherein the compound of formula (IIIa) or a hydrate or pharmaceutically acceptable salt thereof is present in an amount of from about 0.001±0.0002% by weight to about 2.0±0.4% by weight, or from about 0.001±0.0002% by weight to about 1.5±0.3% by weight, or from about 0.001±0.0002% by weight to about 1±0.2% by weight, or from about 0.001±0.0002% by weight to about 0.7±0.14% by weight, or from about 0.001±0.0002% by weight to about 0.5±0.1% by weight, or from about 0.001±0.0002% by weight to about 0.3±0.06% by weight, or from about 0.001±0.0002 wt.% to about 0.2±0.04 wt.%.

A composition according to any of the preceding or following embodiments comprising from about 97% by weight to about 100% by weight of a compound of formula (1a), alternatively from about 98% by weight to 100% by weight, or from about 99% by weight to 100% by weight, based on anhydrous matter.

A composition according to any one of the preceding or following embodiments, wherein the compound of formula (III) is a compound of formula (IV):

-11 049473 ''N

Η

it is o» (IV) (Me-Gly-MePro-Glu), or its stereoisomer, hydrate or pharmaceutically acceptable salt.

A composition according to any one of the preceding or following embodiments, wherein the compound of formula (IV) or a stereoisomer, hydrate, or pharmaceutically acceptable salt thereof is present in an amount of from about 0.001±0.0002% by weight to about 2±0.4% by weight, or from about 0.001±0.0002% by weight to about 1.5±0.3% by weight, or from about 0.001±0.0002% by weight to about 1±0.2% by weight, or from about 0.001±0.0002% by weight to about 0.7±0.14% by weight, or from about 0.001±0.0002% by weight to about 0.5±0.1% by weight, or from about 0.001±0.0002% by weight to about 0.3±0.06% by weight, or from about 0.001±0.0002 wt.% to about 0.2±0.04 wt.%.

A composition according to any one of the preceding or following embodiments, wherein the compound of formula (III) is a compound of formula (V):

Η ⁰¹¹ (V) (Me ₂ -Gly-MePro-Glu), or its stereoisomer, hydrate or pharmaceutically acceptable salt.

A composition according to any one of the preceding or following embodiments, wherein the compound of formula (V) or a stereoisomer, hydrate, or pharmaceutically acceptable salt thereof is present in an amount of from about 0.001±0.0002% by weight to about 2±0.4% by weight, or from about 0.001±0.0002% by weight to about 1.5±0.3% by weight, or from about 0.001±0.0002% by weight to about 1±0.2% by weight, or from about 0.001±0.0002% by weight to about 0.7±0.14% by weight, or from about 0.001±0.0002% by weight to about 0.5±0.1% by weight, or from about 0.001±0.0002% by weight to about 0.3±0.06% by weight, or from about 0.001±0.0002 wt.% to about 0.2±0.04 wt.%.

A composition according to any one of the preceding or following embodiments, wherein the compound of formula (III) is a compound of formula (VI):

Η

χ (VI) (Gly-MePro-Glu(OiPr)-OH), or its stereoisomer, hydrate or pharmaceutically acceptable salt.

A composition according to any one of the preceding or following embodiments, wherein the compound of formula (VI) or a stereoisomer, hydrate, or pharmaceutically acceptable salt thereof is present in an amount of from about 0.001±0.0002% by weight to about 2±0.4% by weight, or from about 0.001±0.0002% by weight to about 1.5±0.3% by weight, or from about 0.001±0.0002% by weight to about 1±0.2% by weight, or from about 0.001±0.0002% by weight to about 0.7±0.14% by weight, or from about 0.001±0.0002% by weight to about 0.5±0.1% by weight, or from about 0.001±0.0002% by weight to about 0.3±0.06% by weight, or from about 0.001±0.0002 wt.% to about 0.2±0.04 wt.%.

-12049473

A composition according to any one of the preceding or following embodiments, wherein the compound of formula (III) is a compound of formula (VII):

CD 11 (VII) (Gly-MePro-Glu-OiPr), or its stereoisomer, hydrate or pharmaceutically acceptable salt.

A composition according to any one of the preceding or following embodiments, wherein the compound of formula (VII) or a stereoisomer, hydrate, or pharmaceutically acceptable salt thereof is present in an amount of from about 0.001±0.0002 wt.% to about 2±0.4 wt.%, or from about 0.001±0.0002 wt.% to about 1.5±0.3 wt.%, or from about 0.001±0.0002 wt.% to about 1±0.2 wt.%, or from about 0.001±0.0002 wt.% to about 0.7±0.14 wt.%, or from about 0.001±0.0002 wt.% to about 0.5±0.1 wt.%, or from about 0.001±0.0002 wt.% to about 0.3±0.06 wt.%, or from about 0.001±0.0002 wt.% to about 0.2±0.04 wt.%.

A composition according to any one of the preceding or following embodiments, wherein the compound of formula (III) is a compound of formula (VIII):

HjN

(VIII) (Gly-MePro-Glu-(OiPr) ₂ ), or its stereoisomer, hydrate or pharmaceutically acceptable salt.

A composition according to any one of the preceding or following embodiments, wherein the compound of formula (VIII) or a stereoisomer, hydrate, or pharmaceutically acceptable salt thereof is present in an amount of from about 0.001±0.0002 wt.% to about 2±0.4 wt.%, or from about 0.001±0.0002 wt.% to about 1.5±0.3 wt.%, or from about 0.001±0.0002 wt.% to about 1±0.2 wt.%, or from about 0.001±0.0002 wt.% to about 0.7±0.14 wt.%, or from about 0.001±0.0002 wt.% to about 0.5±0.1 wt.%, or from about 0.001±0.0002 wt.% to about 0.3±0.06 wt.%, or from about 0.001±0.0002 wt.% to about 0.2±0.04 wt.%.

A composition comprising a compound according to any of the preceding or following embodiments

(1a) (trofinetide), said composition being obtained by:

a) coupling reactions between H-MeP-OH and Z-Gly-OH in the presence of an activating reagent, a silylating agent and a solvent, or

b) coupling reactions between Z-Gly-OH and Suc-OH, and then coupling reactions between the resulting ZGly-OSu and H-MePto-OH in the absence or in the presence of an activating reagent, a solvent, and in the absence or in the presence of a silylating agent;

c) coupling reactions between the obtained Z-Gly-MePro-OH and H-Glu-OH in the presence of an activating reagent, a silylating agent and a solvent;

d) obtaining Z-Gly-MePro-Glu-OH; and

e) removing the protecting group from Z-Gly-MePro-Glu-OH to obtain a composition containing a compound of formula (1a).

A composition according to any of the preceding or following embodiments,

-13 049473 wherein the resulting composition is dissolved or suspended in a solvent.

A composition according to any one of the preceding or following embodiments, wherein said composition further comprises a compound of formula (II):

or a stereoisomer, hydrate or pharmaceutically acceptable salt thereof; and optionally contains a compound of formula (III):

or a stereoisomer, hydrate or pharmaceutically acceptable salt thereof, wherein R _b R ₂ , R3 and R ₄ are independently selected from the group consisting of hydrogen and C] ₄ alkyl, provided that at least one of Ri, R ₂ , R ₃ and R ₄ is C] ₄ alkyl.

A composition according to any of the preceding or following embodiments, wherein the compound of formula (II) or a stereoisomer, hydrate or pharmaceutically acceptable salt thereof is absent or present in an amount of from about 0.001±0.0002 wt.% to about 2±0.4 wt.%; and the compound of formula (III) or a stereoisomer, hydrate or pharmaceutically acceptable salt thereof is absent or present in an amount of from about 0.001±0.0002 wt.% to about 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.

A composition according to any of the preceding or following embodiments, wherein the compound of formula (II) or a stereoisomer, hydrate, or pharmaceutically acceptable salt thereof is present in an amount of from about 0.001±0.0002 wt.% to about 0.3±0.06 wt.%.

A composition according to any of the preceding or following embodiments, wherein the compound of formula (II) or a stereoisomer, hydrate, or pharmaceutically acceptable salt thereof is present in an amount of from about 0.001±0.0002 wt.% to about 0.1±0.02 wt.%.

A composition according to any one of the preceding or following embodiments, wherein the compound of formula (II) or a stereoisomer, hydrate, or pharmaceutically acceptable salt thereof is present in an amount of from about 0.001±0.0002% by weight to about 2±0.4% by weight, or from about 0.001±0.0002% by weight to about 1.5±0.3% by weight, or from about 0.001±0.0002% by weight to about 1±0.2% by weight, or from about 0.001±0.0002% by weight to about 0.7±0.14% by weight, or from about 0.001±0.0002% by weight to about 0.5±0.1% by weight, or from about 0.001±0.0002% by weight to about 0.3±0.06% by weight, or from about 0.001±0.0002 wt.% to about 0.2±0.04 wt.%.

A composition according to any one of the preceding or following embodiments, wherein the compound of formula (II) is a compound of formula (IIa):

^it (IIa)(Z-Gly-MePro-Glu-OH), or its hydrate or pharmaceutically acceptable salt.

A composition according to any of the preceding or following embodiments,

-14049473 wherein the compound of formula (II) or its stereoisomer, hydrate or pharmaceutically acceptable salt is absent.

A composition according to any of the preceding or following embodiments, wherein the compound of formula (III) or a stereoisomer, hydrate or pharmaceutically acceptable salt thereof is present in an amount of from about 0.001±0.0002 wt.% to about 2.0±0.4 wt.%.

A composition according to any of the preceding or following embodiments, wherein said silylating agent does not contain a cyano group.

A composition according to any of the preceding or following embodiments, wherein said silylating 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.

A composition according to any of the preceding or following embodiments, wherein said solvent in step a) is a polar organic solvent or a polar aprotic organic solvent.

A composition according to any of the preceding or following embodiments, wherein said solvent in step b) is a polar organic solvent or a polar aprotic organic solvent.

A composition according to any of the preceding or following embodiments, wherein said solvent in step c) is a polar organic solvent or a polar aprotic organic solvent.

A composition according to any of the preceding or following embodiments, wherein the solvent is selected from the group consisting of alkyl acetates, chlorinated hydrocarbons, alkyl cyanides and amide type solvents, or a mixture thereof.

A composition according to any of the preceding or following embodiments, wherein said activating reagent is selected from the group consisting of carbodiimides, acyl halides, phosphonium salts, uronium salts and guanidinium salts.

A composition according to any of the preceding or following embodiments, wherein the removal of the protecting group is achieved by hydrogenation.

A composition according to any of the preceding or following embodiments, wherein the hydrogenation is carried out in the presence of a Pd/C catalyst.

A composition according to any of the preceding or following embodiments, wherein the hydrogenation is carried out in the presence of a Pd/Si catalyst.

The composition according to any of the preceding or following embodiments, wherein the hydrogenation is carried out at a temperature of about 10-40°C, or at a temperature of about 20-30°C, or at a temperature of about 25°C.

A composition according to any of the preceding or following embodiments, wherein the hydrogenation is carried out at a pressure of from about 0 to about 6 bar.

A composition according to any of the preceding or following embodiments, 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.

A composition according to any of the preceding or following embodiments, comprising at least 90 wt.%, such as at least 91 wt.%, 92 wt.%, 93 wt.%, 94 wt.%, 95 wt.%, 96 wt.% and 97 wt.%, of a compound of formula (I) or a stereoisomer, hydrate or pharmaceutically acceptable salt thereof, based on the anhydrous substance.

A composition according to any of the preceding or following embodiments, comprising at least 90 wt.%, such as at least 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, based on the anhydrous substance.

A composition comprising at least 90 wt.%, such as at least 91 wt.%, 92 wt.%, 93 wt.%, 94 wt.%, 95 wt.%, 96 wt.% and 97 wt.% of a compound of formula (I) or a stereoisomer, hydrate or pharmaceutically acceptable salt thereof, based on the anhydrous substance.

A composition comprising at least 90 wt.%, such as at least 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, based on the anhydrous substance.

A composition according to any of the preceding or following embodiments comprising a compound of formula (I)

- 15 049473

or its stereoisomer, hydrate or pharmaceutically acceptable salt, and a compound of formula (II):

(P), or its stereoisomer, hydrate or pharmaceutically acceptable salt, and/or a compound of formula (III):

r ₄ (III), or a stereoisomer, hydrate or pharmaceutically acceptable salt thereof, wherein R|. R ₂ , R3 and R ₄ are independently selected from the group consisting of hydrogen and C1.4 alkyl, provided that at least one of Ri, R ₂ , R3 and R4 is C1.4 alkyl, and wherein the composition comprises at least 90 wt.%, such as 91 wt.%, 92 wt.%, 93 wt.%, 94 wt.%, 95 wt.%, 96 wt.% or 97 wt.% of the compound of formula (I), calculated on anhydrous basis.

A composition according to any of the preceding or following embodiments comprising a compound of formula (1a)

ⁱⁿ (1a) (trofinetide), or its hydrate or pharmaceutically acceptable salt, and a compound of formula (II): o

(P), or its stereoisomer, hydrate or pharmaceutically acceptable salt, and/or a compound of formula (III):

-16049473

or a stereoisomer, hydrate or pharmaceutically acceptable salt thereof, wherein R|. R ₂ , R ₃ and R ₄ are independently selected from the group consisting of hydrogen and C] ₄ alkyl, provided that at least one of Ri, R ₂ , R ₃ and R | is C] ₄ alkyl, and wherein the composition comprises at least 90 wt.%, such as 91 wt.%, 92 wt.%, 93 wt.%, 94 wt.%, 95 wt.%, 96 wt.% or 97 wt.% of the compound of formula (1a), calculated on an anhydrous basis.

A composition comprising a compound of formula (I)

or its stereoisomer, hydrate or pharmaceutically acceptable salt, and a compound of formula (II):

or its stereoisomer, hydrate or pharmaceutically acceptable salt, and/or a compound of formula (III):

or a stereoisomer, hydrate or pharmaceutically acceptable salt thereof, wherein R|. R ₂ , R ₃ and R ₄ are independently selected from the group consisting of hydrogen and Cj. ₄ alkyl, provided that at least one of Ri, R ₂ , R ₃ and R | is Cj. ₄ alkyl, and wherein the composition comprises at least 90 wt.%, such as 91 wt.%, 92 wt.%, 93 wt.%, 94 wt.%, 95 wt.%, 96 wt.% or 97 wt.% of the compound of formula (I), calculated on an anhydrous basis.

A composition containing a compound of formula (1a)

(1a) (trofinetide), or its hydrate or pharmaceutically acceptable salt, and a compound of formula (II):

- 17049473

or its stereoisomer, hydrate or pharmaceutically acceptable salt, and/or a compound of formula (III):

or a stereoisomer, hydrate or pharmaceutically acceptable salt thereof, wherein R|. R ₂ , R ₃ and R ₄ are independently selected from the group consisting of hydrogen and C] ₄ alkyl, provided that at least one of Ri, R ₂ , R ₃ and Ri is C] ₄ alkyl, and wherein the composition comprises at least 90 wt.%, such as 91 wt.%, 92 wt.%, 93 wt.%, 94 wt.%, 95 wt.%, 96 wt.% or 97 wt.% of the compound of formula (1a), calculated on an anhydrous basis.

A method for producing a composition containing trofinetide according to any of the preceding or following embodiments, comprising the steps of:

a) coupling reactions between H-MePro-OH and Z-Gly-OH in the presence of an activating reagent, a silylating agent and a solvent, or

b) coupling reactions between Z-Gly-OH and Suc-OH, and then coupling reactions between the resulting ZGly-OSu and H-MePrO-OH in the absence or in the presence of an activating reagent, a solvent, and in the absence or in the presence of a silylating agent;

c) coupling reactions between the obtained Z-Gly-MePro-OH and H-Glu-OH in the presence of an activating reagent, a silylating agent and a solvent;

d) obtaining Z-Gly-MePro-Glu-OH; and

e) removing the protecting group from Z-Gly-MePro-Glu-OH, thereby obtaining trofinetide, wherein trofinetide is dissolved or suspended in a solvent.

The method according to any of the preceding or following embodiments, wherein said silylating agent does not contain a cyano group.

The method according to any of the preceding or following embodiments, wherein said silylating 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.

The method according to any of the preceding or following embodiments, wherein said solvent in step a) is a polar organic solvent or a polar aprotic organic solvent.

The method according to any of the preceding or following embodiments, wherein said solvent in step b) is a polar organic solvent or a polar aprotic organic solvent.

The method according to any of the preceding or following embodiments, wherein said solvent in step c) is a polar organic solvent or a polar aprotic organic solvent.

The method according to any of the preceding or following embodiments, wherein the solvent is selected from the group consisting of alkyl acetates, chlorinated hydrocarbons, alkyl cyanides and amide type solvents, or a mixture thereof.

The method according to any of the preceding or following embodiments, wherein said activating reagent is selected from the group consisting of carbodiimides, acyl halides, phosphonium salts, uronium salts and guanidinium salts.

A method according to any of the preceding or following embodiments, wherein

-18049473 Removal of the protective group is achieved by hydrogenation.

The process according to any of the preceding or following embodiments, wherein the hydrogenation is carried out in the presence of a Pd/C catalyst.

The method according to any of the preceding or following embodiments, wherein the hydrogenation is carried out in the presence of a Pd/Si catalyst.

The method according to any of the preceding or following embodiments, wherein the hydrogenation is carried out at a temperature of about 10-40°C, or at a temperature of about 20-30°C, or at a temperature of about 25°C.

The method according to any of the preceding or following embodiments, wherein the hydrogenation is carried out at a pressure of from about 0 to about 6 bar.

The method according to any of the preceding or following embodiments, wherein the hydrogenation is carried out in at least one solvent selected from the group consisting of water, ethyl acetate, isopropyl acetate, methanol, ethanol, isopropanol, or mixtures thereof.

A product comprising a composition according to any of the preceding or following embodiments.

A product according to any of the preceding or following embodiments, wherein the product is a kit.

A kit comprising a dosage form according to any of the preceding or following embodiments, comprising a compound of formula (1a),

(1a) (trofinetide) or its hydrate or pharmaceutically acceptable salt and a compound of formula (Pa):

(Pa) (Z-Gly-MePro-Glu-OH), or a hydrate or a pharmaceutically acceptable salt thereof, and instructions for administration to an individual in need thereof.

A kit according to any of the preceding or following embodiments, further comprising one or more compounds selected from the group consisting of compounds of formula (III), formula (IIIa), formula (IV), formula (V), formula (VI), formula (VII), formula (VIII), and formula (IX).

A product according to any of the preceding or following embodiments, comprising a kit containing a dosage form with instructions for use, containing a compound of formula (1a)

(1a) (trofinetide), or its hydrate or pharmaceutically acceptable salt, and a compound of formula (Pa)

-19049473

^OH (IIa)(Z-Gly-MePro-Glu-OH), or a hydrate or a pharmaceutically acceptable salt thereof, wherein the product contains from 95 wt.% to 105 wt.%, such as 96 wt.%, 97 wt.%, 98 wt.%, 99 wt.%, 100 wt.%, 101 wt.%, 102 wt.%, 103 wt.% or 104 wt.% of a given amount of the compound of formula (1a) in the product.

A product according to any of the preceding embodiments, comprising a kit containing a dosage form with instructions for use, containing a compound of formula (1a):

ⁱⁿ (1a) (trofinetide), or its hydrate or pharmaceutically acceptable salt, and a compound of formula (Pa)

OH on (IIa)(Z-Gly-MePro-Glu-OH), or a hydrate or a pharmaceutically acceptable salt thereof, and further comprising one or more compounds selected from the group consisting of compounds of formula (III), formula (IIIa), formula (IV), formula (V), formula (VI), formula (VII), formula (VIII) and formula (IX), wherein the composition comprises from 95 wt.% to 105 wt.%, such as 96 wt.%, 97 wt.%, 98 wt.%, 99 wt.%, 100 wt.%, 101 wt.%, 102 wt.%, 103 wt.% or 104 wt.% of a given amount of a compound of formula (1a) in the product.

A product comprising a kit containing a dosage form with instructions for use, containing a compound of formula (1a)

(1a) (trofinetide), or its hydrate or pharmaceutically acceptable salt, and a compound of formula (Pa)

he °n (IIa)(Z-Gly-MePro-Glu-OH) or a hydrate or a pharmaceutically acceptable salt thereof, wherein the product contains from 95 wt.% to 105 wt.%, such as 96 wt.%, 97 wt.%, 98 wt.%, 99 wt.%, 100 wt.%, 101 wt.%, 102 wt.%, 103 wt.% or 104 wt.% of a given amount of the compound of formula (1a) in the product.

A product comprising a kit containing a dosage form with instructions for use, containing a compound of formula (1a)

-20049473

(la) (trofinetide), or a hydrate or pharmaceutically acceptable salt thereof, and a compound of formula (Pa)

or a hydrate thereof or a pharmaceutically acceptable ^it (IIa) (Z-Gly-MePro-Glu-OH), salt and further comprising one or more compounds selected from the group consisting of compounds of formula (III), formula (IIIa), formula (IV), formula (V), formula (VI), formula (VII), formula (VIII) and formula (IX), wherein the composition comprises from 95 wt.% to 105 wt.%, such as 96 wt.%, 97 wt.%, 98 wt.%, 99 wt.%, 100 wt.%, 101 wt.%, 102 wt.%, 103 wt.% or 104 wt.% of a given amount of a compound of formula (1a) in the product.

Preparation of compounds and compositions of formula (I)

During the preparation of the compounds of the present invention, compounds are formed having a stereochemistry determined by the reactants and well understood by those skilled in the art. In situations where the reactants have mixed stereochemistry, such as racemates or other permutations of stereoisomers thereof, the resulting compounds will have a correspondingly mixed stereochemistry. In certain embodiments and aspects of the compounds and compositions described herein, stereochemistry is specified, while in others it is not. When the stereochemistry of the compounds is not specified in a formula or chemical structure, it is understood that all possible aspects of stereochemistry are included.

It should be understood that the methods described herein represent specific steps only. Other schemes may be developed to prepare compounds of formula I.

Obtaining trofinetide (formula (1a))

The method for producing a composition containing trofinetide mainly comprises the following steps:

a) coupling reactions between H-MePro-OH and Z-Gly-OH in the presence of an activating reagent, a silylating agent and a solvent, or

b) coupling reactions between Z-Gly-OH and Suc-OH, and then coupling reactions between the resulting ZGly-OSu and H-MePrO-OH in the absence or in the presence of an activating reagent, a solvent, and in the absence or in the presence of a silylating agent;

c) coupling reactions between the obtained Z-Gly-MePro-OH and H-Glu-OH in the presence of an activating reagent, a silylating agent and a solvent;

d) obtaining Z-Gly-MePro-Glu-OH with at least 95% conversion; and

e) removing the protecting group from Z-Gly-MePro-Glu-OH, wherein Z-Gly-MePro-Glu-OH is dissolved or suspended in a solvent, to obtain trofinetide.

These basic stages are described in more detail in the examples below.

Getting other connections

In addition to the compound of formula (I), certain other products may be obtained and may be present in the composition described herein. An example of a compound or stereoisomer of formula (I) is the compound of formula (1a) (trofinetide). Other products include compounds (II), (Ia), (III), (IIIa), (IV), (V), (VI), (VII), (VIII) and (IX), as described above.

Additional products include kits containing a dosage form containing a compound of formula (1a) (trofinetide) and a compound of formula (Pa) and instructions for use.

The kit may further comprise one or more compounds selected from the group consisting of compounds of formula (III), formula (IIIa), formula (IV), formula (V), formula (VI), formula (VII), formula (VHI), and formula (IX).

-21 049473

Examples

The preparation of compositions including trofinstide is illustrated below. It may be appreciated that other schemes may be used to prepare trofinstide.

Example 1: Method for obtaining trofinetide

In general, trofinetide and related compounds can be prepared from a peptide or amino acid precursor that is reacted with a silylating agent or persilylation agent in one or more steps. Silylating agents such as N-trialkylsilylamines or N-trialkylsilylamides that do not contain a cyano group can be used within the scope of the present invention.

Examples of such silylating reagents 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, 3(trimethylsilyl)-2-oxazolidone.

Step 1: Obtaining Z-Gly-OSu

Several alternative techniques can be used for this stage. Technique 1A

1. SucOH

2. EDC.HCI

2-Gly-OH

Bright formula: C. _1c H , ₁ N O ₊ formula*: 209.19864 iPrOH

Z-Gly-OSu

Molecular formula: CuH^Nj-Og Mass by formula: 306.27076

One (1) equiv of Z-Gly-OH and 1.1 equiv of Suc-OH were solubilized in 27 equiv of iPrOH and 4 equiv _{of CH2Cl2} at 21 °C. The mixture was cooled and when the temperature reached -4 °C, 1.1 equiv of EDC.HC1 was gradually added maintaining the temperature below 10 °C. A dense solid appeared during the reaction. After the addition of EDC.HC1, the mixture was allowed to warm up to 20 °C. The suspension was cooled to 11 °C and filtered. The filter cake was washed with 4.9 equiv of cold iPrOH and 11 equiv of IPE and then dried at 34 °C (dried product Z-Gly-OSu - purity: 99.5%; NMR analysis: 96%; yield: 84%).

Method 1B

This procedure is a variation of Procedure 1A, differing in the replacement of iPrOH with ACN. One (1) equiv of Z-Gly-OH and 1.1 equiv of Suc-OH were solubilized in 22 equiv in ACN at 35 °C. The mixture was cooled in an ice bath. When the temperature reached 1 °C, 0.9 equiv of DCC in 5.5 equiv of ACN was gradually added to maintain the temperature below 5 °C. The coupling reaction was allowed to proceed for approximately 20 h. During the course of the reaction, DCU precipitated and was removed by filtration at the end of the coupling. After filtration, the DCU was washed with ACN to isolate the product. The Z-Gly-OSu mixture was then concentrated to achieve 60% by weight. iPrOH (17 equiv) was added to initiate crystallization. A dense solid appeared shortly after the addition of iPrOH. An additional 17 equiv of iPrOH was required to liquefy the suspension. The suspension was cooled in an ice bath and filtered. The solid was washed with 9 equiv. iPrOH and then dried at 45°C (dried product Z-Gly-OSu - purity: 99.2%; HPLC analysis: 99.6%; yield: 71%).

Step 2: Obtaining Z-Gly-MePro-OH

Several alternative techniques can be used for this stage.

Method 2A

MePro.HCI ZSIyHePro

S-vukyarkm f&rtsuga. S _N -Μ.ΰ. Myachtyarymfrv^ta: Ο,Η-.ΝΟι, Mshkvugafiaya foryuga: S.,N.1 ₃ O_

Mass according to formula: Mass according to formula: 125.15704 Mass according to formula: 320.34-04

One (1) equiv of MePro.HCl _was partially solubilized in 29 equiv of _CH2Cl2 at 35 °C with 1.04 equiv of TEA and 1.6 equiv of TMA. The mixture was heated at 35 °C for 2 h to effect silylation. Then 1.02 equiv of Z-Gly-OSu was added to the mixture. The mixture was maintained at 35 °C for 3 h and then 0.075 equiv of butylamine was added to quench the reaction. The mixture was allowed to return to room temperature and stirred for at least 15 min. Z-Gly-MePro-OH was extracted once with 5% w/w _NaHCO3 in 186 equiv of water, then three times sequentially with 5% w/w _NaHCO3 in 62 equiv of water. The aqueous layers were combined and the pH was adjusted to 2.2 by adding 34 equiv H2O as 12 N H2O at room temperature. At this pH, Z-Gly-MePro-OH formed a thick solid that was solubilized at 45° with approximately 33 equiv EtOAc and 2.3 equiv iButOH. Z-Gly-MePro-OH was extracted into or

-22049473 organic layer and washed with 62 equiv. of demineralized water. Then the organic layer was dried by azeotropic distillation with 11.5 equiv. of EtOAc until the peptide started to precipitate. Cyclohexane (12 equiv.) was added to the mixture to complete the precipitation. The suspension was cooled at 5 °C for 2 h and filtered. The solid was washed with 10 equiv. of cyclohexane and then dried at 45 °C (dried product Z-Gly-MePro-OH - purity: 100%; HPLC analysis: 100%; yield 79%).

Method 2B

This procedure is a variation of Procedure 2A. One (1) equiv. MePro.HCl _was partially solubilized in 36.6 equiv. _CH2Cl2 at 34 °C with 1.01 equiv. TEA and 0.1 equiv. TMA. Then 1.05 equiv. ZGly-OSu was added to the mixture, followed by 1.0 equiv. TEA. The mixture was maintained at 35 °C for approximately 1 h, cooled to 25-30 °C and 0.075 equiv. DMAPA was added to stop the reaction. One hundred (100) equiv. water, 8.6 equiv. HCl as 12 N HCl and 0.3 equiv. _KHSO4 were added to the mixture (no precipitation was observed, pH = 1.7). ZGly-MePro-OH was extracted into the organic layer and washed twice with 97 equiv. of demineralized water with 0.3 equiv. _{of KHSO4} , then 100 equiv. of demineralized water, respectively. EtOAc (23 equiv.) was added to the mixture and _CH2Cl2 was _removed by distillation until the peptide began to precipitate. Cyclohexane (25 equiv.) was added to the mixture to complete the precipitation. The suspension was cooled at 2 °C overnight and filtered. The solid was washed with 21 equiv. of cyclohexane and then dried at 39 °C (dried product Z-Gly-MePro-OH - purity: 98.7%; NMR analysis: 98%; yield 86%).

Method 2 C

Pyridine. DIPEA, Ριν-Cl

2. MePro.HCl DIPEA _: TMA

EUAs

In reactor 1, MePro.HCl (1 equiv) was suspended in EtOAc (approximately 7 equiv). DIPEA (1 equiv) and TMA (2 equiv) were added and the mixture was heated to dissolve the solids. Once dissolved, the solution was cooled to 0 °C. In reactor 2, Z-Gly-OH (1 equiv) was suspended in EtOAc (approximately 15 equiv). DIPEA (1 equiv) and pyridine (1 equiv) were added. After stirring, the solution was obtained and cooled to -5 °C. Piv-Cl (1 equiv) was added to reactor 2 and the contents of reactor 1 were added to reactor 2. After complete addition, the contents of reactor 2 were brought to room temperature. The conversion of Z-Gly-OH to Z-Gly-MePro-OH was monitored by HPLC. When the reaction was complete, the reaction mixture was quenched with DMAPA (0.1 equiv.) and washed with an aqueous solution containing _KHSO4 (ca. 2.5 wt.%), NaCl (ca. 4 wt.%), and conc. HCl (ca. 6 wt.%) in 100 equiv. _H2O . The aqueous layer was re-extracted with EtOAc and the combined organic layers were washed with an aqueous solution containing _KHSO4 (ca. 2.5 wt.%) and NaCl (ca. 2.5 wt.%) in 100 equiv. _H2O , followed by water (100 equiv.). Residual water was removed from the organic Z-GlyMePro-OH solution by vacuum distillation with EtOAc. The resulting suspension was diluted with heptane (ca. 15 equiv.) and cooled to 0 °C. The product was isolated by filtration, washed with cold heptane (approximately 7 equiv.) and dried in vacuo at 45°C. Z-Gly-MePro-OH was obtained (yield 85%).

Step 3: Obtaining Z-Gly-MePro-Glu-OH

Several alternative techniques can be used at this stage.

Methodology FOR

Glu

Molecular formula: CH _L NOL· Mass by formula: 147.1232?

H-Glu-OH (1.05 equiv) was silylated in 2 equiv _{CH2Cl2 with} 3.5 equiv TMA at 65 °C. Silylation was complete after 2 h. While silylation was occurring, 1.0 equiv Z-Gly-MePro-OH and 1.0 equiv Oxyma Pure were solubilized in 24 equiv _CH2Cl2 and 1.0 equiv DMA at room temperature in another reactor. EDC.HC1 (1.0 equiv) was added. The activation level reached 97% after 15 min. Then _the activated Oxyma Pure solution was added to the silylated H-Glu-OH at 40 °C and cooled at room temperature. The coupling reaction time was approximately 15 min, with the coupling level reaching 97%. Addition of 8.2% w/w IaHCO3 in 156 equiv. water to the mixture at room temperature (with _CO2 evolution) was performed to achieve pH 8. Z-Gly-MePro-Glu-OH extracts

-23 049473 was dissolved in water. The aqueous layer was washed twice _with 29 equiv. _CH2Cl2 . Residual _CH2Cl2 was removed by concentration. The pH was adjusted to 2.5 with 2.5 N HCl and then 1.4 equiv. _of solid _KHSO4 was added to precipitate Z-Gly-MePro-Glu-OH. The mixture was filtered and the solid was washed with 3x52 equiv. water. The filtered solid was added to 311 equiv. of demineralized water and heated to 55-60 °C. iPrOH (29 equiv.) was added gradually until the product was completely solubilized. The mixture was slowly cooled to 10 °C with moderate stirring for 40 min to initiate crystallization. The peptide was filtered and washed with 2x52 equiv. water and then dried at 45°C (dried product Z-Gly-MePro-Glu-OH - purity: 99.5%; NMR analysis: 96%; yield 74%).

Method 3B

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

3C Methodology

This procedure was a variation of Procedure 3A. H-Glu-OH (1.05 equiv) was silylated in 3.7 equiv _CH2Cl2 with 3.5 equiv TMA at 62°C. Silylation was complete in approximately 1.5-2 _h as evidenced by solubilization. During the silylation step, 1.0 equiv Z-Gly-MePro-OH and 1.0 equiv Oxyma Pure were solubilized in 31.5 _{equiv CH2Cl2} at 22°C. One (1.06) equiv EDC.HCl was added to complete the activation. The silylated H-Glu-OH was then added to the activated Oxyma Pure solution. The temperature was controlled during the addition to remain below 45°C. Desilylation was performed by adding a mixture of 2.5% w/w KHSO4 in 153 equiv. water and 9 equiv. iPrOH to achieve pH 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 and the solid was washed with 90 equiv. water and then dried at 36 °C.

3D Methodology

This procedure is a variation of Procedure 3A. H-Glu-OH (1.05 equiv) _was silylated in 3.9 equiv _CH2Cl2 with 3.5 equiv TMA at 62°C. Silylation was complete after 2 h as evidenced by solubilization. During the silylation step, 1 equiv Z-Gly-MePro-OH and 1 equiv Oxyma Pure were solubilized in 25 equiv _CH2Cl2 at ₂₃ °C. One (1) equiv EDC.HCl was added. An additional 0.07 equiv EDC.HCl was added to complete the reaction. Silylated H-Glu-OH was then added to the activated Oxyma Pure solution. The temperature was controlled during the addition to remain below 45°C. Desilylation was performed by adding a mixture of 2.5% w/w KHSO4 in 160 equiv water and 9.6 equiv iPrOH to achieve pH 1.63.

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 equiv. water and then dried at approximately 25 °C for 2.5 days. The solid was then solubilized at 64 °C by adding 55 equiv. water and 31 equiv. iPrOH. After solubilization, the mixture was diluted with 275 equiv. water and cooled to 10 °C to crystallize. The mixture was filtered and the solid was washed with 60 equiv. water and then dried at 27 °C (dried product Z-Gly-MePro-Glu-OH - purity: 99.6%; NMR analysis: 98%; yield 74%).

- 24 049473

ZE Methodology

In reactor 1, H-Glu-OH (1.05 equiv) was suspended in ACN (approximately 2.2 equiv). TMA (approximately 3.5 equiv) was added and the mixture was heated to dissolve the solids. Once dissolved, the solution was cooled to room temperature. In reactor 2, Z-Gly-MePro-OH (1 equiv) was suspended in ACN (14 equiv). Oxyma Pure (1 equiv) and EDC.HC1 (1 equiv) were added. The mixture was stirred at room temperature until the solids were dissolved. The contents of reactor 2 were added to reactor 1. The conversion of Z-Gly-MePro-OH to Z-Gly-MePro-Glu-OH was monitored by HPLC. After completion, the reaction mixture was added to an aqueous solution containing _KHSO4 (approximately 2.5 wt%) dissolved in approximately 100 equiv. _H2O . ACN was removed from the aqueous suspension of Z-Gly-MePro-Glu-OH by vacuum distillation with _H2O . After stirring at room temperature, the product in the resulting suspension was isolated by filtration and washed with water. The resulting solid was dissolved in an aqueous solution containing NaHCO (approximately 5 wt%) in 11 equiv. _H2O and recrystallized by adding an aqueous solution containing _KHSO4 (approximately 10 wt%) in 90 equiv. _H2O . The product was isolated by filtration, washed with water and dried in vacuo at 45°C. Z-Gly-MePro-Glu-OH was obtained (yield 75%).

Step 4: Removal of the protecting group and release of trophinetide

Several alternative techniques can be used at this stage.

Method 4A

Z-Gly-MePro-Glu-OH (1 equiv) was suspended in water (approximately 25 equiv) and EtOAc (approximately 15 equiv). Pd/C (0.025 equiv by weight and containing 10% Pd by weight) was added and the reaction mixture was hydrogenated by bubbling hydrogen through the reaction mixture at room temperature. Conversion of Z-Gly-MePro-Glu-OH to trofinetide was monitored by HPLC and after completion of the reaction the catalyst was removed by filtration and the layers were separated. Residual EtOAc 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. Trofinetide was obtained (yield 90%). Alternatively, deprotection can be accomplished using MeOH alone or a combination of iPrOH and MeOH, or using ethyl acetate in water.

Method 4B

This procedure is a variation of procedure 4A, except for EtOAc. Z-Gly-MePro-Glu-OH (1 equiv) was suspended in water (approximately 50 equiv). Pd/C (0.05 equiv, 5% Pd by weight) was added and the reaction mixture was hydrogenated at room temperature under 5 bar. Conversion of Z-Gly-MePro-Glu-OH to trofinetide was monitored by HPLC. After completion of the reaction, the catalyst was removed by filtration and the aqueous layer was washed with EtOAc (approximately 5 equiv). Residual EtOAc 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. Trofinetide was obtained (90% yield).

Method 4 C

This procedure is a variation of procedure 4A with MeOH replacing EtOAc. Z-Gly-MePro-Glu-OH (1 equiv) was suspended in MeOH (100 equiv) and water (12 equiv). Pd/Si (0.02 equiv w/w) was added and the mixture was heated at 23 C for hydrogenolysis. During deprotection, the peptide was solubilized. Conversion of Z-Gly-MePro-Glu-OH to trophinetide was monitored by HPLC and upon completion of the reaction, the catalyst was removed by filtration and the layers were washed with MeOH and iPrOH. The solvents were concentrated in vacuo at 45 °C and trophinetide precipitated. The precipitate was filtered and dried at 45 °C to afford trophinetide.

-25 049473

4D Methodology

This procedure is a variation of procedure 4A with Pd/C replaced by Pd/Si. One (1.0) equiv of Z-GlyMePro-Glu-OH was partially solubilized in 105 equiv of MeOH and 12 equiv of water. Pd/Si (0.02 equiv by weight) was added and the mixture was heated at 23 °C for hydrogenolysis. The peptide was solubilized during deprotection. At the end of deprotection (approximately 99% conversion after 1 h), the catalyst was filtered and washed with 20-30 equiv of MeOH. iPrOH (93 equiv) was added and MeOH was replaced by iPrOH by concentration at 45 °C under vacuum. The peptide was concentrated until it began to precipitate. The peptide was filtered and dried at 45°C (dried product H-Gly-MePro-Glu-OH: purity: 98.1%; NMR analysis: 90%; yield 81%).

Methodology 4E

This procedure is a variation of procedure 4A with removal of _H2O and replacement of Pd/C with Pd/Si. One (1.0) equiv of Z-Gly-MePro-Glu-OH was partially solubilized in 44 equiv of MeOH. Pd/Si type 340 (0.02 equiv by weight) was added and the mixture was maintained at 20 °C for hydrogenolysis. The peptide was solubilized during deprotection. At the end of deprotection (approximately 99.9% conversion, after 3-3.5 h), the catalyst was filtered off and washed with 8 equiv of MeOH. The deprotected peptide was then precipitated in 56 equiv of iPrOH. After 30 min at 5 °C, the peptide was filtered off and washed three times with 11 equiv of HCl. iPrOH and then dried at 25°C (dried product H-Gly-MePro-Glu-OH: purity: 99.4%; HPLC analysis: ~98%; yield: 81%).

4F Methodology

This procedure is a variation of Procedure 4A. One (1) equiv of Z-Gly-MePro-Glu-OH was partially solubilized in 14 equiv of EtOAc and 25 equiv of water. Pd/C (0.01 equiv w/w) was added and the mixture was maintained at 20 °C for hydrogenolysis. The peptide was solubilized during deprotection. At the end of deprotection (approximately 100% conversion, after approximately 3.5 h), the catalyst was filtered off and washed with a mixture of 3.5 equiv of EtOAc and 6 equiv of water. The aqueous layer was then ready for spray drying (aqueous solution of H-Gly-MePro-Glu-OH peptide: purity: 98.6%; yield: ~95%).

4G Methodology

This procedure is a variant of procedure 4A with Pd/C replaced by Pd/Si, EtOAc by MeOH, and _H2O removed. Pd/Si type 340 (0.02 equiv w/w) was added to 2.9 vol MeOH for pre-reduction for 30 min. One (1.0) equiv of Z-Gly-MePro-Glu-OH was partially solubilized in 34 equiv MeOH. The reduced palladium was then transferred to the peptide mixture. The mixture was maintained at 20 °C for hydrogenolysis. During deprotection, the peptide was solubilized. Pd/C type 39 (0.007 equiv w/w) was added to the mixture to enhance the reaction kinetics. At the end of deprotection, the catalyst was filtered and washed with 13.6 equiv MeOH. The deprotected peptide was then precipitated in 71 equiv iPrOH. After approximately 40 min the peptide was filtered and washed with 35 equiv iPrOH. The peptide was dried below 20°C and was then ready for solubilization in water and spray drying.

4H Methodology

This procedure is a variation of Procedure 4A. One (1.0) equiv of Z-Gly-MePro-Glu-OH was partially solubilized in 24.8 equiv of water and 13.6 equiv of EtOAc. Pd/C type 39 (0.025 equiv by weight) was added to the peptide mixture. The mixture was maintained at 20 °C for hydrogenolysis. The peptide was solubilized during deprotection. At the end of deprotection (19 h), the catalyst was removed by filtration and washed with 5.3 equiv of water and 2.9 equiv of EtOAc. The biphasic mixture was then decanted to remove the upper organic layer. The aqueous layer was diluted with water to achieve a concentration of HGly-MePro-Glu-OH suitable for spray drying the solution.

Example 2: Alternative method for obtaining trofinetide

An alternative method for synthesizing trofinetide is based on US Patent No. 8,546,530, adapted for the tripeptide, as follows.

The persilylated compounds used to synthesize the compound of formula (Ia) (trofinetide) are obtained by silylation of the corresponding peptide or amino acid by reaction with a silylating agent, optionally in an organic solvent. If desired, the persilylated peptide or amino acid can be isolated and purified. It is possible to use the persilylated peptide or amino acid in situ, for example, by combining a solution containing the persilylated peptide or amino acid with a solution containing an optionally activated peptide or amino acid.

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

In step 3, the persilylated amino acid compound is obtained by silylation of the corresponding

- 26 049473 the relevant amino acid (e.g. H-Glu-OH) by reaction with a silylating agent, optionally in an organic solvent. If desired, the persilylated amino acid or peptide can be isolated and purified. However, it is suitable to use the persilylated amino acid or peptide in situ, for example by combining a solution containing the persilylated amino acid with a solution containing an optionally activated (e.g. using EDC.HCl and Oxyma Pure) peptide (e.g. Z-Gly-MePro-OH).

In the context of the present invention, it is suitable to use silylating agents such as N-trialkylsilylamines or N-trialkylsilylamides that do not contain a cyano group. Examples of such silylating reagents 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, 3-(trimethylsilyl)-2-oxazolidone.

The reaction of step 2 is typically carried out at a temperature of from 0°C to 100°C, optionally from 10°C to 40°C, and optionally from 15°C to 30°C.

The reaction of step 3 is typically carried out at a temperature of from 0°C to 100°C, optionally from 10°C to 60°C, optionally from 15°C to 50°C.

In the reaction of step 2, generally from 0.5 to 5 equivalents, optionally from 1 to 3 equivalents, optionally about 1.5 to 2.5 equivalents of the silylating agent are used relative to the molar amount of the functional groups to be silylated. It is also possible to use from 2 to 4 equivalents of the silylating agent relative to the molar amount of the functional groups to be silylated. The expression functional groups to be silylated means specific groups having an active hydrogen atom that can react with the silylating agent, such as amino, hydroxyl, mercapto or carboxyl groups.

In step 3 of the reaction, generally from 0.5 to 5 equivalents, optionally from 2 to 4.5 equivalents, optionally about 3 to 4 equivalents of the silylating agent relative to the molar amount of the functional groups to be silylated are used. It is also possible to use from 2.5 to 4.5 equivalents of the silylating agent relative to the molar amount of the functional groups to be silylated.

It is understood that persilylated means an amino acid or a peptide or an amino acid analogue or a peptide analogue in which the groups having an active hydrogen atom that can react with the silylating agent are sufficiently silylated to ensure that a uniform reaction medium is obtained for the coupling reaction step.

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

Good results are often achieved by using additional carboxyl group activating reagents that reduce side reactions and/or increase the efficiency of the reaction. For example, phosphonium and uronium salts can, in the presence of a tertiary base such as N,N-diisopropylethylamine (DIPEA) and triethylamine (TEA), convert protected amino acids to activated forms. Other reagents help prevent racemization by providing a protecting reagent. These reagents include carbodiimides (e.g., DCC) with an additional auxiliary nucleophile (e.g., 1-hydroxybenzotriazole (HOBt), 1-hydroxyazabenzotriazole (HOAt), or Suc-OH) or derivatives thereof. Another reagent that can be used is TBTU. The mixed anhydride route using isobutyl chloroformate with or without an additional auxiliary nucleophile is also used, as is the azide route, due to the low racemization associated with it. These types of compounds can also enhance the rate of carbodiimide-mediated addition. Typical additional reagents also include bases such as N,N-diisopropylethylamine (DIPEA), triethylamine (TEA), or Nmethylmorpholine (NMM).

When the silylation is carried out in the presence of a solvent, said solvent is optionally a polar organic solvent, even more particularly, optionally, a polar aprotic organic solvent. An amide type solvent such as N,N-dimethylformamide (DMF) or N,N-dimethylacetamide (DMAC) can be used. In the context of the present invention, an alkyl acetate solvent can be used for step 2, in particular, a more particular optional embodiment is ethyl acetate.

In the context of the present invention, a chlorinated hydrocarbon solvent or an alkyl cyanide solvent can be used for step 3, in particular, a more specifically optional

- 27 049473 the option is dichloromethane or acetonitrile.

In another embodiment, the silylation is carried out in a liquid silylation medium consisting essentially of a silylation agent and an amino acid or peptide.

Within the context of the present invention, an amino acid or a peptide is understood to refer in particular to an amino acid or a peptide or an amino acid analogue or a peptide analogue which are linked at their N-terminus or optionally at another position to the carboxyl group of an amino acid or a peptide with a protected amino group.

Example 3: Descriptions of compositions containing compounds of formula (I)___________

Analysis Description Appearance Powder from white to off-white Identification according to LC RRT 1.00+/-0.02 vs. reference standard LC analysis on a dry matter basis 97% - 100% w/w Total amount of impurities NMT 2% w/w Single unidentified impurities <0.05% w/w Single identified impurities <0.05% or qualification level (% w/w) Z-Gly-MePro-Glu-OH <0.3% w/w Water content NMT 7.5% w/w Residual solvents Complies with ICH Q3C ¹

¹ ICH Q3C Guideline on Impurities: Guidance on Residual Solvents

Example 4: Alternative Procedure 4B of Step 4 of Example 1 for the Preparation of Trofinetide

This procedure is a variation of procedure 4B, step 4. Z-Gly-MePro-Glu-OH (1 equiv) was added portionwise to Pd/C (0.027 equiv w/w and containing 5% Pd w/w) in approximately 50 equiv water. The reaction mixture was hydrogenated at 20 °C under 5 bar for at least 4 cycles of 4 h each. Between cycles, Pd/C (0.0027 equiv w/w) was added as needed to accelerate the reaction. Conversion of Z-Gly-MePro-Glu-OH to trophinetide was monitored by HPLC. After completion of the reaction, the catalyst was removed by filtration, washed with water (12.5 equiv) and the aqueous layer was washed with EtOAc (approximately 14 equiv). After phase separation, residual EIAc was removed from the aqueous solution containing trofinetide by bubbling nitrogen under vacuum at 20°C for approximately 3 h. The aqueous solution was filtered. The final concentration of trofinetide was approximately 25 wt% and the solution was then ready for spray drying to isolate the product.

Example 5: Alternative formulation of trofinetide Composition comprising a compound of formula (I)

or its stereoisomer, hydrate or pharmaceutically acceptable salt, and a compound of formula (II):

or its stereoisomer, hydrate or pharmaceutically acceptable salt, and/or a compound of formula (ΠΙ):

-28049473

' (ΠΙ), or a stereoisomer, hydrate or pharmaceutically acceptable salt thereof, wherein R|. R ₂ , R ₃ and R ₄ are independently selected from the group consisting of hydrogen and C] ₄ alkyl, provided that at least one of Ri, R ₂ , R3 and R | is C] ₄ alkyl, and wherein the composition comprises at least 90 wt.%, such as 91 wt.%, 92 wt.%, 93 wt.%, 94 wt.%, 95 wt.%, 96 wt.% or 97 wt.% of a compound of formula (I), calculated on an anhydrous basis.

Example 6: Alternative formulation of trofinetide

A composition containing a compound of formula (1a)

(1a) (trofinetide), or its hydrate, or a pharmaceutically acceptable salt, and a compound of formula (II):

it (P), or its stereoisomer, hydrate or pharmaceutically acceptable salt, and/or a compound of formula (III):

R

or a stereoisomer, hydrate or pharmaceutically acceptable salt thereof, wherein R|. R ₂ , R3 and R ₄ are independently selected from the group consisting of hydrogen and C] ₄ alkyl, provided that at least one of Ri, R ₂ , R3 and R| is C] ₄ alkyl, and wherein the composition comprises at least 90 wt.%, such as 91 wt.%, 92 wt.%, 93 wt.%, 94 wt.%, 95 wt.%, 96 wt.% or 97 wt.% of the compound of formula (1a), calculated on an anhydrous basis.

Example 7: Trofinetide Product

A product comprising a kit containing a dosage form with instructions for use containing a compound of formula (1a)

OH (1a) (trofinetide), or its hydrate, or a pharmaceutically acceptable salt, and a compound of formula (Pa)

-29049473

^it (Pa) (Z-Gly-MePro-Glu-OH), or a hydrate or a pharmaceutically acceptable salt thereof, wherein the product contains from 95 wt.% to 105 wt.%, such as 96 wt.%, 97 wt.%, 98 wt.%, 99 wt.%, 100 wt.%, 101 wt.%, 102 wt.%, 103 wt.% or 104 wt.% of a given amount of the compound of formula (1a) in the product.

Example 8: Trofinetide Product

A product comprising a kit containing a dosage form with instructions for use, containing a compound of formula (1a)

(1a) (trofinetide), or its hydrate or pharmaceutically acceptable salt, and a compound of formula (Pa) o g—A

^it (Pa) (Z-Gly-MePro-Glu-OH), or a hydrate or a pharmaceutically acceptable salt thereof, and further comprising one or more compounds selected from the group consisting of compounds of formula (III), formula (IIIa), formula (IV), formula (V), formula (VI), formula (VII), formula (VIII) and formula (IX), wherein the composition comprises from 95 wt.% to 105 wt.%, such as 96 wt.%, 97 wt.%, 98 wt.%, 99 wt.%, 100 wt.%, 101 wt.%, 102 wt.%, 103 wt.%, or 104 wt.% of a given amount of compound of formula (1a) in the product.

Example 9: Analysis of products and compositions

The products and compositions described herein can be analyzed by liquid chromatography, a suitable chromatographic method using UHPLC, for example using materials and conditions such as Waters Acquity CSH C18, 1.7 μm, column 150 x 2.1 mm, water with 0.1% TFA (mobile phase A), and water/ACN 70/30+0.1% TFA (mobile phase B), in the range (from 4% phase A/6% phase B to 100% phase B and washing with 4% phase A/6% phase B). Flow rate: 0.35 ml/min, column temperature: 40 °C, autosampler temperature: 4 °C, injection volume: 4 μl (for example, obtained by weighing approximately 10 mg of powder into a 10 ml volumetric flask and diluting to the required volume with water). Examples of detectors are UV (ultraviolet, UV 220 nm) and MS (mass spectrometry).

Industrial applicability

The present invention is applicable in pharmaceutical, medical and other health care fields.

Claims (20)

CLAUSE OF THE INVENTION 1. A pharmaceutical composition containing a compound of formula (I) or a stereoisomer, hydrate or pharmaceutically acceptable salt thereof, and from about 0.001 wt.% to about 2 wt.% of a compound of formula (II): or its stereoisomer, hydrate or pharmaceutically acceptable salt. 2. The composition of claim 1, wherein the compound of formula (II) or a stereoisomer, hydrate or pharmaceutically acceptable salt thereof is present in an amount of from about 0.001 wt.% to about 1 wt.%. 3. The composition according to claim 1 or 2, wherein the compound of formula (II) or a stereoisomer, hydrate or pharmaceutically acceptable salt thereof is present in an amount of from about 0.001 wt.% to about 0.5 wt.%. 4. The composition according to any one of claims 1 to 3, wherein the compound of formula (II) or a stereoisomer, hydrate or pharmaceutically acceptable salt thereof is present in an amount of from about 0.001 wt.% to about 0.3 wt.%. 5. The composition according to any one of claims 1 to 4, wherein the compound of formula (II) or a stereoisomer, hydrate or pharmaceutically acceptable salt thereof is present in an amount of from about 0.001 wt.% to about 0.2 wt.%. 6. The composition according to any one of claims 1 to 5, wherein the compound of formula (II) or a stereoisomer, hydrate or pharmaceutically acceptable salt thereof is present in an amount of from about 0.001 wt.% to about 0.1 wt.%. 7. A composition according to any one of claims 1 to 6, wherein the compound of formula (I) is a compound of formula (Ia) or its hydrate or pharmaceutically acceptable salt. 8. A composition according to claim 7, containing a compound of formula (Ia) in the form of an anhydrous substance. 9. A composition according to any one of claims 1 to 8, wherein the compound of formula (II) is a compound of formula (IIa): - 31 049473 "" (Pa), or its hydrate or pharmaceutically acceptable salt. 10. A composition according to claim 7 or 8, comprising a compound of formula (Ia), wherein said composition is obtained by: a) a coupling reaction between (28)-2-methylpyrrolidine-2-carboxylic acid and benzyloxycarbonylglycine in the presence of an activating reagent, a silylating agent and a solvent, or b) a coupling reaction between benzyloxycarbonylglycine and N-hydroxysuccinimide, followed by a coupling reaction between the resulting N-succinimidyl ester of benzyloxycarbonylglycine and (28)-2-methylpyrrolidine-2-carboxylic acid in the absence or presence of an activating reagent, a solvent, and in the absence or presence of a silylating agent; c) coupling reactions between the obtained ((8)-1-(((benzyloxy)carbonyl)glycyl)-2-methylpyrrolidine-2-carboxylic acid) and (28)-2-aminopentanedioic acid in the presence of an activating reagent, a silylating agent and a solvent; d) obtaining ((S)-1-(((benzyloxy)carbonyl)glycyl)-2-methylpyrrolidine-2-carbonyl)-L-glutamic acid; and e) removing the protecting group from (^)-1-(((benzyloxy)carbonyl)glycyl)-2-methylpyrrolidine-2-carbonyl)-L-glutamic acid to obtain a composition containing a compound of formula (Ia). 11. The composition according to claim 10, wherein the compound of formula (II) is a compound of formula (IIa): ^0.1 (Pa), or its hydrate or pharmaceutically acceptable salt. 12. The composition of claim 11, wherein the compound of formula (IIa) or a stereoisomer, hydrate or pharmaceutically acceptable salt thereof is present in an amount of from about 0.001 wt.% to about 1 wt.%. 13. The composition of claim 11 or 12, wherein the compound of formula (IIa) or a stereoisomer, hydrate or pharmaceutically acceptable salt thereof is present in an amount of from about 0.001 wt.% to about 0.5 wt.%. 14. The composition of any one of claims 11 to 13, wherein the compound of formula (IIa) or a stereoisomer, hydrate or pharmaceutically acceptable salt thereof is present in an amount of from about 0.001 wt.% to about 0.3 wt.%. 15. The composition of any one of claims 11 to 14, wherein the compound of formula (IIa) or a stereoisomer, hydrate or pharmaceutically acceptable salt thereof is present in an amount of from about 0.001 wt.% to about 0.1 wt.%. 16. The composition according to any one of claims 10 to 15, wherein the removal of the protective group is achieved by hydrogenation. 17. The composition according to claim 16, wherein the hydrogenation is carried out in the presence of a Pd/C catalyst. 18. The composition according to claim 16, wherein the hydrogenation is carried out in the presence of a Pd/Si catalyst. 19. The composition according to any one of claims 16 to 18, 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. 20. A pharmaceutical composition containing a compound of formula (Ia) - 32 049473 or a hydrate or pharmaceutically acceptable salt thereof, and from about 0.001 wt.% to about 0.2 wt.% of a compound of formula (IIa): or its hydrate or pharmaceutically acceptable salt. Evra Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky per., 2 -