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WO2024166022A1 - Procédé de préparation d'un fragment tétrapeptidique de liraglutide - Google Patents

Procédé de préparation d'un fragment tétrapeptidique de liraglutide Download PDF

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Publication number
WO2024166022A1
WO2024166022A1 PCT/IB2024/051149 IB2024051149W WO2024166022A1 WO 2024166022 A1 WO2024166022 A1 WO 2024166022A1 IB 2024051149 W IB2024051149 W IB 2024051149W WO 2024166022 A1 WO2024166022 A1 WO 2024166022A1
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Prior art keywords
formula
group
process according
hydroxy
tetrapeptide
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PCT/IB2024/051149
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English (en)
Inventor
Nitin Kumar
Abhishek GIRI
Hrishikesh Dilip Acharya
Govind Singh
Walter Cabri
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Fresenius Kabi Oncology Ltd
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Fresenius Kabi Oncology Ltd
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Priority to KR1020257029895A priority Critical patent/KR20250148640A/ko
Priority to EP24710827.7A priority patent/EP4662222A1/fr
Priority to CN202480011717.8A priority patent/CN120659800A/zh
Publication of WO2024166022A1 publication Critical patent/WO2024166022A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1024—Tetrapeptides with the first amino acid being heterocyclic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons

Definitions

  • the present invention relates to an improved process for the preparation of a tetrapeptide of formula I, used in the synthesis of Liraglutide.
  • Ri and Rs are independently selected from an amine protecting group, and Rs is t-Bu or Bn.
  • the present invention further, relates to a process for the preparation of Liraglutide or its pharmaceutically acceptable salts, using the tetrapeptide of formula I prepared by the process of the present invention.
  • Liraglutide a human GLP-1 receptor agonist (or GLP-1 analogue) is represented by the formula,
  • Liraglutide sold under the brand name Victoza ® (Novo Nordisk), is an anti-diabetic medication used to treat type 2 diabetes, obesity, and chronic weight management. It was approved for medical use in the European Union in 2009, and in the United States in 2010.
  • Liraglutide is prepared by chemical synthesis, either via solid phase synthesis or liquid phase by sequential coupling of amino acids, or by a convergent synthesis involving coupling of fragments, which were synthesised separately.
  • Most of the processes known in the art for synthesis of Liraglutide involve synthesis of various short chain peptide fragments such as a dipeptide, a tripeptide, a tetrapeptide, a pentapeptide and the like, either in solution or solid phase. These fragments are then coupled either in solid or solution phase or via a hybrid approach, to provide Liraglutide.
  • the tetrapeptide fragment of formula I is one such fragment, which is used in the synthesis of Liraglutide.
  • WO2016046753A1 discloses a process for the synthesis of the tetrapeptide fragment where it is prepared by a solid phase synthesis with a purity of 94% by HPLC.
  • a solid phase synthesis of tetrapeptide of formula I is also disclosed in CN102875665B and CN106478805B with a purity of about 94% and 96.6% by HPLC respectively.
  • CN105732798B discloses another process for the synthesis of the tetrapeptide fragment of Formula I.
  • the disclosed process involves a linear coupling of histidine (His) with alanine (Ala), glutamic acid (Glu) and glycine (Gly) in presence of a solvent and a base.
  • the tetrapeptide of formula I contains histidine at the amino terminal. Histidine is an amino acid that is highly prone to racemization. On exposure to base, the tendency of histidine towards racemization increases.
  • the present invention relates to an improved process for the preparation of tetrapeptide of formula I, used in the synthesis of Liraglutide.
  • the present invention relates to an improved process for the preparation of a tetrapeptide of formula I, Formula I wherein Ri and Rs are independently selected from an amine protecting group, and Rs is t-Bu or Bn, comprising the step of condensing an activated compound of formula II,
  • the present invention relates to a process for the preparation of Liraglutide or its pharmaceutically acceptable salts thereof, comprising converting the tetrapeptide of formula I, obtained by the process of the present invention, to Liraglutide or its pharmaceutically acceptable salts thereof.
  • “Peptide” refers to a short chain of amino acids, wherein two or more amino acids are chemically linked by an amide linkage.
  • Diapeptide refers to a peptide having a chain of two amino acids.
  • Tripeptide refers to a peptide having a chain of three amino acids.
  • Tetrapeptide ’ refers to a peptide having a chain of four amino acids.
  • fragment refers to a sequence of two or more amino acids present.
  • the amino acids in the fragment may be protected or unprotected.
  • condensation refers to a condensation reaction where an amide linkage is formed by reaction of a carboxylic group with an amine group.
  • carboxylic acid refers to an organic compound that contains a -COOH group.
  • amine refers to an organic compound that contains a -NH2 group.
  • activated compound refers to a compound with an activated ester group.
  • acid activating group refers to a group that enhances the reactivity of a carboxylic group. An activated carboxylic acid undergoes the same reactions as their inactivated analogues but do so more rapidly.
  • activated ester refers to an ester functional group that is highly susceptible towards nucleophilic attack. An activated ester undergoes the same reactions as their inactivated analogues but do so more rapidly. An activated ester can be prepared by conversion of the hydroxyl (-OH) of the carboxylic acid group into a good leaving group by reaction with an activating agent.
  • leaving group refers to an atom, a group of atoms or to a fragment that detaches from the main or residual part of a substrate during a reaction or elementary step of a reaction.
  • activating agent refers to a compound that causes activation of a molecule or a group towards reaction with another group or a molecule by donating an activating group into the molecule.
  • Coupled reagent refers to a reagent that facilitates formation of a bond between two adjacent groups.
  • protecting group refers to a temporarily attached group to a functional group to decrease the reactivity of the functional group so that the protected functional group does not react under synthetic conditions to which the molecule is subjected in one or more subsequent steps, at the same time allowing for removal of such protecting group under conditions, which do not harm the remaining molecule.
  • amine protecting group specifically refers to a protecting group that is attached to an amine functionality in a molecule.
  • an “amine protecting group ” in a molecule having two or more amine protecting groups may be the same or different from one another.
  • the protecting groups can be cleaved from the molecule after the desired compound is obtained.
  • the term “deprotecting” refers to the cleavage or removal of the protecting groups.
  • the protecting group can be deprotected under acidic, basic and/or neutral conditions.
  • the protection and deprotection methods are well known in the art (see notably "Protective groups in organic synthesis”, Greene T. W. and Wuts P. G. M., Wiley-Interscience, 1999).
  • ambient temperature refers to a temperature ranging from about 15°C to 35°C.
  • the present invention relates to a process for the preparation of a tetrapeptide of formula I, wherein Ri and Rs are independently selected from an amine protecting group, Rs is t-Bu or Bn.
  • the amine protecting group is selected from the group consisting of tertbutyloxycarbonyl, trityl, 4-methyltrityl, monomethoxytrityl, fluorenylmethyloxycarbonyl, carboxybenzyl, N-benzyloxymethyl and toluenesulfonyl.
  • Ri and R2 are the same amine protecting groups.
  • Ri and R2 are different amine protecting groups.
  • Ri is selected from tert-butyloxycarbonyl, trityl, 4-methyltrityl, monomethoxytrityl, carboxybenzyl, fluorenylmethyloxycarbonyl.
  • R2 is selected from trityl, 4-methyltrityl, monomethoxytrityl, N- benzyloxymethyl, fluorenylmethyloxycarbonyl, toluenesulfonyl and tert-butyloxy carbonyl.
  • Ri is tert-butyloxycarbonyl and R2 is selected from trityl and 4- methyltrityl.
  • Ri is tert-butyloxycarbonyl and R2 is trityl.
  • the process comprises a step of condensing an activated compound of formula II, wherein Ri and R2are as defined above; A is an acid activating group, with a tripeptide of formula III,
  • the acid activating group A in compound of formula II is the ester group introduced from the activating agents selected from N-hydroxy succinimide, N-hydroxy-5-norbomene-2,3- dicarboximide, 1 -hydroxybenzotriazole, 6-chloro-l- hydroxybenzotriazole, l-hydroxy-7- azabenzotriazole, 3-hydroxy-4-oxo-3,4-dihydro-l,2,3-benzotriazine, ethyl 1 -hydroxy- 1H- 1,2,3- triazole-4-carboxylate, and N-hydroxytetrazole.
  • the activating agents selected from N-hydroxy succinimide, N-hydroxy-5-norbomene-2,3- dicarboximide, 1 -hydroxybenzotriazole, 6-chloro-l- hydroxybenzotriazole, l-hydroxy-7- azabenzotriazole, 3-hydroxy-4-oxo-3,4-dihydro-l,2,3-
  • condensation reaction of the compound of formula II and the compound of formula III is carried out in presence of a base and a solvent.
  • the base can be selected from the group consisting of N, N-diisopropylethylamine, triethylamine, methyl morpholine, sodium bicarbonate, sodium carbonate and potassium carbonate, preferably triethylamine.
  • the solvent can be selected from the group consisting of dichloromethane, 1 -methyl -pyrrolidin-2- one, acetonitrile, tetrahydrofuran, dimethyl sulfoxide, N, N-dimethyl formamide, N-methyl- formamide, dimethyl carbonate, diethyl carbonate and a mixture thereof.
  • the solvent is dichloromethane .
  • the condensation reaction is carried out at an ambient temperature, preferably at a temperature of 20°C to 35 °C, more preferably at a temperature of 20°C to 30°C.
  • the tetrapeptide of formula I is a compound of Formula la [Formula I, wherein Ri is Boc, R2 is Trt, R3 is t-Bu] .
  • the present invention relates to a process of preparation of a compound of formula la comprising condensing an activated compound of formula Ila [Formula II, wherein Ri is Boc and R2 is Trt and A is ONB],
  • condensation is carried out in presence of dichloromethane and triethylamine at a temperature of 25 °C to 30°C .
  • the reaction mixture is stirred for 2-4 hours and the tetrapeptide of formula la is isolated from the reaction mixture after extraction with dichloromethane and precipitation with ethyl acetate.
  • the activated compound of formula II is prepared by activating the carboxylic group of a compound of formula IV,
  • the activating agent can be selected from the group consisting of N-hydroxysuccinimide, N-hydroxy- 5-norbomene-2,3-dicarboximide, 1 -hydroxybenzotriazole, 6-chloro-l- hydroxybenzotriazole, 1- hydroxy-7-azabenzotriazole, 3-hydroxy-4-oxo-3,4-dihydro-l,2,3-benzotriazine, ethyl 1-hydroxy- IH-l,2,3-triazole-4-carboxylate and N-hydroxytetrazole.
  • the activating agent is N- hydroxy-5 -norbomene -2,3 -dicarboximide .
  • the activation of the carboxylic group is carried out in the presence of a coupling reagent in a solvent.
  • the coupling reagent can be selected from the group consisting of N-(3-dimethylaminopropyl)-N'- ethylcarbodiimide hydrochloride, N, N-dicyclohexylcarbodiimide, Oxyma B /diisopropyl carbodiimide, benzotriazole- 1 -yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate, hexafluorophosphate azabenzotriazole tetramethyl uranium, O-(lH-benzotriazol-l-yl)-N,N,N',N'- tetramethyluronium hexafluorophosphate, O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate and propylphosphonic anhydride.
  • the coupling agent is selected from the group consisting of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, N, N- dicyclohexylcarbodiimide and Oxyma B /diisopropyl carbodiimide. More preferably, the coupling agent is N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride.
  • the solvent can be selected from the group consisting of dichloromethane, 1 -methyl -pyrrolidin-2- one, acetonitrile, tetrahydrofuran, dimethyl sulfoxide, N, N-dimethyl formamide, N-methyl- formamide, dimethyl carbonate, diethyl carbonate and a mixture thereof.
  • the activation of the carboxylic group with an activating agent is carried out in the presence of a coupling reagent in tetrahydrofuran.
  • the compound of Formula IV wherein Ri is Boc and R2 is Trt, is activated with N-hydroxy-5-norbomene-2,3-dicarboximide (HONB) to form activated compound of formula IIa[Formula II, wherein Ri is Boc and R2 is Trt and A is ONB] .
  • HONB N-hydroxy-5-norbomene-2,3-dicarboximide
  • the compound of Formula IV is treated with N-hydroxy-5- norbomene-2,3-dicarboximide (HONB) in tetrahydrofuran in presence of N-(3- dimethylaminopropyl)-N'-ethyl carbodiimide hydrochloride at 20-30°C temperature for 10 to 12 hours.
  • HONB N-hydroxy-5- norbomene-2,3-dicarboximide
  • the activated compound of formula II is isolated after the completion of reaction by addition of water and dichloromethane.
  • the present invention relates to process for the preparation of a tripeptide of formula III comprising a step of activating the carboxylic group of a compound of formula V,
  • the amine protecting group (R4) can be selected from fluorenylmethyloxycarbonyl, tertbutyloxycarbonyl, carboxybenzyl and toluene sulfonyl, preferably it is fluorenylmethyloxycarbonyl.
  • the activating agent can be selected from the group consisting of N-hydroxysuccinimide, N-hydroxy- 5-norbomene-2,3-dicarboximide, 1 -hydroxybenzotriazole, 6-chloro-l- hydroxybenzotriazole, 1- hydroxy-7-azabenzotriazole, 3-hydroxy-4-oxo-3,4-dihydro-l,2,3-benzotriazine, ethyl 1-hydroxy- lH-l,2,3-triazole-4-carboxylate and N-hydroxytetrazole.
  • the activating agent is N- hydroxy-5 -norbomene -2,3 -dicarboximide .
  • the activation of the carboxylic group is carried out in the presence of a coupling reagent in a solvent.
  • the coupling reagent can be selected from the group consisting of N-(3-dimethylaminopropyl)-N'- ethylcarbodiimide hydrochloride, N, N-dicyclohexylcarbodiimide, Oxyma B /diisopropyl carbodiimide, benzotriazole- 1 -yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate, hexafluorophosphate azabenzotriazole tetramethyl uronium, O-(lH-benzotriazol-l-yl)-N,N,N',N'- tetramethyluronium hexafluorophosphate, O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate and propylphosphonic anhydride.
  • the coupling agent is selected from the group consisting of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, N, N- dicyclohexylcarbodiimide and Oxyma B /diisopropyl carbodiimide. More preferably, the coupling agent is N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride.
  • the solvent can be selected from the group consisting of dichloromethane, 1 -methyl -pyrrolidin-2- one, acetonitrile, tetrahydrofuran, dimethyl sulfoxide, N, N-dimethyl formamide, N-methyl- formamide, dimethyl carbonate, diethyl carbonate and a mixture thereof.
  • the activation of the carboxylic group with an activating agent is carried out in the presence of a coupling reagent in tetrahydrofuran.
  • the compound of formula V, wherein R4 is Fmoc is activated with N- hydroxy-5-norbomene-2,3-dicarboximide (HONB) to give activated compound of formula VI , wherein R4 is Fmoc; A is ONB.
  • HONB N- hydroxy-5-norbomene-2,3-dicarboximide
  • the compound of formula V is treated with N-hydroxy-5- norbomene-2,3-dicarboximide (HONB) in tetrahydrofuran in presence of N-(3- dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride at 25°C to 30°C for 10 to 12 hours to give the activated compound of formula VI.
  • the activated compound of formula VI is isolated in dichloromethane after the completion of reaction by addition of water and dichloromethane.
  • the activated compound of formula VI is further condensed with a dipeptide of formula VII,
  • the amine protecting group (R4) can be selected from fluorenylmethyloxycarbonyl, tertbutyloxycarbonyl, carboxybenzyl and toluene sulfonyl, preferably it is fluorenylmethyloxycarbonyl.
  • the base can be selected from the group consisting of N, N-diisopropylethylamine, triethylamine, methyl morpholine, sodium bicarbonate, sodium carbonate and potassium carbonate.
  • the base is triethylamine.
  • the solvent can be selected from the group consisting of dichloromethane, 1 -methyl -pyrrolidin-2- one, acetonitrile, tetrahydrofuran, dimethyl sulfoxide, N, N-dimethyl formamide, N-methyl- formamide, dimethyl carbonate, diethyl carbonate and a mixture thereof.
  • the solvent is dichloromethane .
  • the activated compound of formula VI, wherein R4 is Fmoc; A is ONB, is condensed with the dipeptide of formula VII, wherein R3 is t-Bu, to provide a protected tripeptide of formula VIII, wherein R3 is t-Bu, R4 is Fmoc.
  • condensation is carried out in the presence of triethylamine and dichloromethane as solvent.
  • the tripeptide of formula III is obtained by deprotecting the protected tripeptide of formula VIII by any of the known methods in the art.
  • the deprotection is performed either in an acid or a base, depending on the protecting group to be removed.
  • the protected tripeptide of formula VIII is deprotected in presence of an organic base in a solvent.
  • the organic base can be selected from the group consisting of ammonia, diethylamine, piperidine, piperazine, tributylamine, pyrrolidine, ethanolamine, morpholine, 1,8 -diazabicyclo [5.4.0] undec-7- ene, 1,4-diazabicyclo [2.2. 2]octane, and dicyclohexylamine.
  • the organic base is diethylamine.
  • the solvent in the deprotection reaction can be selected from the group consisting of dichloromethane, 1 -methyl -pyrrolidin-2 -one, acetonitrile, tetrahydrofuran, dimethyl sulfoxide, N, N- dimethyl formamide, N-methyl-formamide, dimethyl carbonate, diethyl carbonate and a mixture thereof.
  • this solvent is dichloromethane.
  • the deprotection of the protected tripeptide of Formula VIII can be carried out in the presence diethylamine in dichloromethane solvent.
  • the deprotection is carried out at a temperature of 30°C to 50°C, more preferably at a temperature of 35°C to 45°C.
  • the solid can be isolated from the reaction mixture by any suitable method. In some embodiments, the solid is isolated by additions of diisopropyl ether, filtration and drying.
  • the dipeptide of formula VII is obtained by a process comprising a step of activating the carboxylic group of a compound of formula IX,
  • the amine protecting group R5 can be selected from the group consisting of fluorenylmethyloxycarbonyl, tert-butyloxycarbonyl, carboxybenzyl and toluenesulfonyl; preferably it is fluorenylmethyloxycarbonyl.
  • the activation of formula IX is done by an activating agent in presence of a coupling reagent and a solvent.
  • the activating agent can be selected from the group consisting of N-hydroxysuccinimide, N-hydroxy- 5-norbomene-2,3-dicarboximide, 1 -hydroxybenzotriazole, 6-chloro-l- hydroxybenzotriazole, 1- hydroxy-7-azabenzotriazole, 3-hydroxy-4-oxo-3,4-dihydro-l,2,3-benzotriazine, ethyl 1-hydroxy- lH-l,2,3-triazole-4-carboxylate and N-hydroxytetrazole.
  • the activating agent is N- hydroxy-5 -norbomene -2,3 -dicarboximide .
  • the activation of the carboxylic group is carried out in the presence of a coupling reagent in a solvent.
  • the coupling reagent can be selected from the group consisting of N-(3-dimethylaminopropyl)-N'- ethylcarbodiimide hydrochloride, N, N-dicyclohexylcarbodiimide, Oxyma B /diisopropyl carbodiimide, benzotriazole- 1 -yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate, hexafluorophosphate azabenzotriazole tetramethyl uronium, O-(lH-benzotriazol-l-yl)-N,N,N',N'- tetramethyluronium hexafluorophosphate, O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate and propylphosphonic anhydride.
  • the coupling agent is selected from the group consisting of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, N, N- dicyclohexylcarbodiimide and Oxyma B /diisopropyl carbodiimide. More preferably, the coupling agent is N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride.
  • the solvent can be selected from the group consisting of dichloromethane, 1 -methyl -pyrrolidin-2- one, acetonitrile, tetrahydrofuran, dimethyl sulfoxide, N, N-dimethyl formamide, N-methyl- formamide, dimethyl carbonate, diethyl carbonate and a mixture thereof.
  • the activation of the carboxylic group with an activating agent is carried out in the presence of a coupling reagent in tetrahydrofuran.
  • the compound of formula IX, wherein R5 is Fmoc is activated with N- hydroxy-5-norbomene-2,3-dicarboximide (HONB) to give activated compound of formula X, wherein R5 is Fmoc; A is ONB.
  • HONB N- hydroxy-5-norbomene-2,3-dicarboximide
  • the compound of formula IX is treated with N -hydroxy-5 -norbomene- 2,3-dicarboximide (HONB) in tetrahydrofuran in presence of N-(3-dimethylaminopropyl)-N'- ethylcarbodiimide hydrochloride at 20-30°C for 6 to 8 hours, resulting in the activation of the compound of formula IX.
  • the compound of formula X, so obtained, is extracted in dichloromethane after the completion of reaction by addition of water and dichloromethane.
  • the activated compound of formula X is then condensed with Glycine (Gly) to provide a protected dipeptide which after deprotection yields the dipeptide of formula VII.
  • the condensation of the compound of formula X with Glycine is done in presence of a solvent, preferably dichloromethane and a base, preferably triethylamine.
  • the dipeptide of formula VII is obtained by deprotecting the protected dipeptide obtained above by any of the known methods in the art.
  • the deprotection is performed either in an acid or a base, depending on the protecting group to be removed.
  • the protected dipeptide is reacted with an organic base in a solvent.
  • the organic base is selected from the group consisting of ammonia, diethylamine, piperidine, piperazine, tributylamine, pyrrolidine, ethanolamine, morpholine, 1,8 -diazabicyclo [5.4.0] undec-7- ene, 1,4-diazabicyclo [2.2. 2]octane, and dicyclohexylamine.
  • the organic base is diethylamine.
  • the solvent used for the deprotection reaction in the presence of organic base can be selected from the group consisting of dichloromethane, 1 -methyl -pyrrolidin-2 -one, acetonitrile, tetrahydrofuran, dimethyl sulfoxide, N, N-dimethyl formamide, N-methyl-formamide, dimethyl carbonate, diethyl carbonate and a mixture thereof.
  • the solvent is dichloromethane.
  • the deprotection is carried out in the presence of diethylamine in dichloromethane solvent.
  • the deprotection is carried out at 20-30°C for 5 to 8 hours.
  • the solid can be isolated from the reaction mixture by any suitable method. In some embodiments, the solid is isolated by addition of water and dichloromethane followed by fdtration and drying to yield the dipeptide of formula VII.
  • the activated ester compounds of formula II, VI and X obtained in the process of present invention are prepared, in-situ and are not isolated during the process.
  • the tetrapeptide of formula I is prepared by using histidine in the last stage of the reaction sequence. This restricts the exposure of histidine to the base during the process. Using the process of the present application, the exposure of the histidine residue/histidine containing peptide to the base occurs only once during the synthesis and hence, racemization of histidine to D-isomer is minimised.
  • the inventors have found that by following the sequence of reaction steps and performing the condensation of amino acids with fragments in the order according to the process of the present application, e.g., introducing histidine at the last stage, the impurity of D-isomer of histidine in the tetrapeptide fragment is highly reduced.
  • the prior art process instead discloses introducing histidine at the first stage, thereby exposing histidine residue to base, at every stage during subsequent reaction steps until the formation of the tetrapeptide is completed. This results in racemisation of histidine during the process and thereby yields the product with a high amount of impurity, specifically with a high amount of D-histidine impurity.
  • the inventors of the present invention have also tried the synthesis of tetrapeptide of formula I by introducing histidine at second stage instead of the first stage to reduce the exposure of histidine to base during the process. This also resulted in increased racemisation of histidine leading to D-His impurity of about 3%, whereas the process of the present invention yields the tetrapeptide fragment with negligible amount of D-isomer of histidine, without any further purification.
  • the tetrapeptide of formula I obtained by the process of present invention contains less than 0.5% of D-isomeric impurity of histidine by HPLC, preferably less than 0.4% of D-isomeric impurity of histidine by HPLC, more preferably less than 0.3% of D-isomeric impurity of histidine by HPLC, and even more preferably less than 0.2% of D-isomeric impurity of histidine by HPLC.
  • the process according to the invention results in a tetrapeptide of formula I with a very low amount of D-isomer of histidine, impurity, (e.g., less than 0.5% by HPLC) even in the crude tetrapeptide.
  • crude product may be purified using processes known in the art to remove unreacted compounds (e.g., amino acids).
  • the purification of crude tetrapeptide is carried out to remove the unreacted starting amino acids only and is not required for removing D-His impurity which is already controlled in the crude product obtained by employing the process of the present invention.
  • the tetrapeptide of formula I is purified with a solvent selected from the group consisting of ethyl acetate, acetone, isopropyl alcohol, tetrahydrofuran, acetonitrile and mixtures thereof.
  • the tetrapeptide of formula I obtained by the process of according to the invention has a purity of 98% by HPLC or more, preferably 99% by HPLC or more, most preferably 99.4% by HPLC.
  • the process according to the invention is carried out in solution phase.
  • This also eliminates the disadvantages of solid phase synthesis such as use of costly resins, multiple washing with solvents, and problems of scale up of batches.
  • the tetrapeptide of formula I, obtained by a process of the present invention may be converted to Liraglutide or its pharmaceutically acceptable salts by the methods known in the art, for example, using a process as reported in WO2016046753A1.
  • the invention provides liraglutide or its pharmaceutically acceptable salt thereof which contains less than 0.5% of D-isomeric impurity of histidine by HPLC, preferably less than 0.4% of D-isomeric impurity of histidine by HPLC, more preferably less than 0.3% of D-isomeric impurity of histidine by HPLC, and even more preferably less than 0.2% of D-isomeric impurity of histidine by HPLC.
  • the inventors of the present invention have developed an improved process, not only for the synthesis of the tetrapeptide fragment but also for Liraglutide or its pharmaceutically acceptable salts, which is both cost efficient and commercially viable.
  • the process can be scaled up effectively and the product or its intermediates at various stages of synthesis can be isolated using isolation techniques such as solvent extraction and solvent recovery, precipitation, distillation, filtration and drying of the product.
  • the synthetic method of the present invention thus, improves peptide purity, reduces material and purification cost and is advantageous for industrial production.
  • HONB N-hydroxy-5-norbomene-2,3-dicarboximide, accordingly ONB refers to the same molecule when attached to a second molecule via the hydroxy group, therefore without H at the hydroxy group.
  • Trt Trityl
  • Stage-1 Preparation of Dipeptide: Glu(OtBu)-Gly-OH a) EDC.HC1 (101.4 g) was added to a mixture of HONB (94.78 g) and Fmoc-Glu (OtBu)-OH (150 g) in tetrahydrofuran (1400 ml). The mixture was stirred at 20-30°C for 15-16 hours. Distilled water (750 ml) and brine solution (150 ml) was added into the reaction mixture and stirred for 190 minutes and allowed to settle. The aqueous layer was separated and discarded. Water (750 ml) and brine solution (150 ml) was added to the organic layer. The mixture was stirred.
  • the aqueous layer was discarded.
  • the organic layer was washed with sodium bicarbonate solution (70 gm in 1000 ml water). The layers were separated, and the aqueous layer was discarded. Distilled water was added to the organic layer, and the pH was adjusted to 3.5 with hydrochloric acid. The layers were separated, and the aqueous layer was discarded.
  • the organic layer was washed with distilled water and distilled at 40-45°C and degassed. Acetonitrile (500 ml) was added to the resulting mass and heated up to 40-50°C for 30 min. and then cooled to 35-40 °C. Dichloromethane (50 ml) was added to the reaction mixture and stirred for 1 hour and then cooled to 20-25°C.
  • the reaction mass was evaporated under vacuum at 35-40°C and the obtained residue was diluted with Ethyl Acetate (150 ml). Water was added to the diluted mass and the pH was adjusted to 3.4-3.5 with hydrochloric acid solution. The organic layer was separated and washed with saturated NaHCO3 solution (100ml). Water was added to the organic layer the pH was adjusted to 4.0-4. 1 with hydrochloric acid solution. The organic layer was separated and evaporated under vacuum at 35-40°C. Water and dichloromethane (50 ml) was added to the reaction mass and stirred. The dichloromethane layer was separated and slowly added to diisopropyl ether (150 ml). The precipitated mass was filtered, washed with di isopropyl ether and dried under vacuum at to obtain a solid (6g).
  • Stage-1 Preparation of Dipeptide: Glu(OtBu)-Gly-OH a) EDC.
  • HC1 (101.4 g) was added to a mixture of HONB (94.78 g) and Fmoc-Glu (OtBu)-OH (150 g) in tetrahydrofuran (1400 ml). The mixture was stirred at 20-30°C for 15-16 hours.
  • Distilled water (750 ml) and brine solution (150 ml) was added into the reaction mixture and stirred for 190 minutes and allowed to settle. The aqueous layer was separated and discarded. Water (750 ml) and brine solution (150 ml) was added to the organic layer. The mixture was stirred.
  • the aqueous layer was distilled under vacuum at a temperature below 55°C.
  • dichloromethane 300 ml was charged and was recovered under vacuum at a temperature below 45 °C.
  • the residual mass was degassed under vacuum for 1 to 2 hours at 35 to-45°C.
  • the solid (44.5g) was isolated by addition of dichloromethane followed by filtration, washing with dichloromethane and drying under vacuum at a temperature 40- 50°C for 14-16 hours.
  • Stage -2 Preparation of Tripeptide: Ala-Glu (OtBu)-Gly-OH a) EDC.
  • HC1 ((92.40 g) was added to a mixture of HONB (86.32g) and Fmoc-Ala (100 g) in tetrahydrofuran (500 mL) and was stirred at 20-30°C for 10 to 12 hours.
  • distilled water and dichloromethane 500 ml was added into the reaction mixture and stirred for 10-15 minutes and allowed to settle. The aqueous layer was separated, and the product was extracted in dichloromethane solvent. The dichloromethane layers were combined, and water was added.
  • the solid was isolated from the reaction mixture by fdtration, washings with water and extractions with methanol at a temperature of 35 to 45°C and cooling to 20-30°C.c)
  • Dichloromethane (500 ml) and diethylamine (100 ml) was added to solid (Fmoc-Ala-Glu(OtBu)-Gly-OH) obtained at step b), at 20-30°C .
  • the reaction mass was stirred for 5-8 hours, and distilled water was added. The layers were separated, and the aqueous layer was recovered under vacuum below 50°C.
  • Dichloromethane (500 ml) was added to the above residue and the mixture was heated to 35-45°C.
  • the solid (71g) was isolated from the reaction mixture by additions of diisopropyl ether, filtration and drying under vacuum at 40-50°C for 15-18 hours.
  • Stage-3 Preparation of tetrapeptide: (Boc)-His (Trt)-Ala-Glu(OtBu)-Gly-OH) a) EDC.
  • HC1 (57.8 g) was added to a mixture of HONB ((54.01 g) and Boc-His (Trt)-OH (100 g) in tetrahydrofuran (500 mL). The mixture was stirred at 20-30°C for 10 to 12 hours. After completion of the reaction, distilled water and dichloromethane (500 ml) was added to the reaction mixture and stirred for 10-15 minutes and allowed to settle.
  • Stage-1 Preparation of Dipeptide: Glu(OtBu)-Gly-OH a) EDC. The mixture was stirred at 20-30 °C for 12-15 hours. The reaction mixture was cooled to 10- 15 °C. Pre-cooled distilled water (1000 mL) and cooled brine solution are added into the reaction mixture and stirred. The layers were allowed to settle, separated and the aqueous layer was discarded. The organic layer was washed with pre-cooled distilled water (1000 mL) and cooled brine solution. The mixture was again allowed to settle, and the aqueous layer was discarded. b) To the organic layer, glycine solution and triethylamine solution was added at 10-20 °C.
  • reaction mixture was stirred for 4 -6 hours, and the reaction mixture was cooled to 10- 15 °C.
  • Pre- cooled distilled water (800 mL) and pre-cooled ethyl acetate (2000 mL) was added to it at 10 -15 °C.
  • the pH was adjusted to 3 to 3.5.
  • the reaction mixture was stirred for 10-15 minutes at 10-15 °C, the layers were settled, and the aqueous layer was discarded.
  • pre-cooled sodium bicarbonate solution and cooled brine solution was added at 10-20 °C into the reaction mixture.
  • the reaction mixture was stirred, and the layers were separated.
  • pre-cooled distilled water (1000 mL) was added at 10-15 °C.
  • Dichloromethane (400 mL) was added to it and then recovered under vacuum at below 40 °C temperature.
  • Dichloromethane (400 mL) was again added, and the residual mass was heated to 30-40 °C.
  • Diisopropylether (400 mL) was added, the reaction mass was stirred for 60 minutes and cooled to 20-30 °C and further stirred for 2-3 hours.
  • the solid (46 g) was isolated by fdtration, washing with diisopropylether and drying under vacuum.
  • Stage-3 Preparation of tetrapeptide: (Boc)-His (Trt)-Ala-Glu(OtBu)-Gly-OH) a) EDC.
  • HC1 34.52 g
  • HONB 32.25 g
  • Boc-His (Trt)-OH 60 g
  • tetrahydrofuran 480 mL
  • the mixture was stirred at 25-30 °C for 12- 15 hours and cooled to 10-15 °C.
  • Pre-cooled distilled water (300 mL) and cooled brine solution was simultaneously added into the reaction mixture and stirred. The layers were settled and separated, and the aqueous layer was discarded.
  • the organic layer was washed with distilled water and brine solution, recovered under vacuum at below 50 °C temperature and degassed to obtain a residue.

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Abstract

La présente invention concerne un procédé amélioré pour la préparation d'un tétrapeptide représenté par la formule I, dans laquelle R1 et R2 sont indépendamment choisis parmi un groupe protecteur d'amine et R3 désigne t-Bu ou Bn. La présente invention concerne en outre un procédé de préparation de liraglutide ou de ses sels pharmaceutiquement acceptables faisant appel au tétrapeptide de formule I.
PCT/IB2024/051149 2023-02-10 2024-02-08 Procédé de préparation d'un fragment tétrapeptidique de liraglutide Ceased WO2024166022A1 (fr)

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KR1020257029895A KR20250148640A (ko) 2023-02-10 2024-02-08 리라글루타이드의 테트라펩타이드 단편의 제조 방법
EP24710827.7A EP4662222A1 (fr) 2023-02-10 2024-02-08 Procédé de préparation d'un fragment tétrapeptidique de liraglutide
CN202480011717.8A CN120659800A (zh) 2023-02-10 2024-02-08 制备利拉鲁肽的四肽片段的方法

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6268343B1 (en) 1996-08-30 2001-07-31 Novo Nordisk A/S Derivatives of GLP-1 analogs
CN102875665A (zh) 2012-09-28 2013-01-16 深圳翰宇药业股份有限公司 一种合成利拉鲁肽的方法
WO2016046753A1 (fr) 2014-09-23 2016-03-31 Novetide, Ltd. Synthèse de peptides glp-1
WO2016067271A1 (fr) * 2014-10-31 2016-05-06 Auro Peptides Ltd Procédé de préparation de liraglutide
CN105732798A (zh) 2015-11-03 2016-07-06 江苏诺泰生物制药股份有限公司 一种利拉鲁肽的合成方法
CN106478805A (zh) 2015-08-28 2017-03-08 甘李药业股份有限公司 一种glp-1衍生物的制备方法
WO2021152622A1 (fr) * 2020-01-27 2021-08-05 Neuland Laboratories Limited Procédé amélioré pour la préparation de liraglutide

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6268343B1 (en) 1996-08-30 2001-07-31 Novo Nordisk A/S Derivatives of GLP-1 analogs
CN102875665A (zh) 2012-09-28 2013-01-16 深圳翰宇药业股份有限公司 一种合成利拉鲁肽的方法
WO2016046753A1 (fr) 2014-09-23 2016-03-31 Novetide, Ltd. Synthèse de peptides glp-1
US20180057558A1 (en) * 2014-09-23 2018-03-01 Novetide, Ltd. Synthesis of GLP-1 Peptides
WO2016067271A1 (fr) * 2014-10-31 2016-05-06 Auro Peptides Ltd Procédé de préparation de liraglutide
CN106478805A (zh) 2015-08-28 2017-03-08 甘李药业股份有限公司 一种glp-1衍生物的制备方法
CN105732798A (zh) 2015-11-03 2016-07-06 江苏诺泰生物制药股份有限公司 一种利拉鲁肽的合成方法
WO2021152622A1 (fr) * 2020-01-27 2021-08-05 Neuland Laboratories Limited Procédé amélioré pour la préparation de liraglutide

Non-Patent Citations (1)

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Title
GREENE T. WWUTS P. G. M: "Protective groups in organic synthesis", 1999, WILEY-INTERSCIENCE

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