CN120659800A

CN120659800A - Method for preparing tetrapeptide fragment of liraglutide

Info

Publication number: CN120659800A
Application number: CN202480011717.8A
Authority: CN
Inventors: N·库玛; A·吉里; H·D·阿查里亚; G·辛格; W·卡布里
Original assignee: Fresenius Kabi Oncology Ltd
Current assignee: Fresenius Kabi Oncology Ltd
Priority date: 2023-02-10
Filing date: 2024-02-08
Publication date: 2025-09-16
Also published as: WO2024166022A1; KR20250148640A; EP4662222A1

Abstract

本发明涉及一种用于制备式(I)的四肽的改进方法，其中R₁和R₂独立地选自胺保护基团，R₃为t‑Bu或Bn。本发明进一步涉及一种使用式(I)的四肽制备利拉鲁肽或其药学上可接受盐的方法。 The present invention relates to an improved method for preparing a tetrapeptide of formula (I), wherein _R1 and _R2 are independently selected from amine protecting groups and _R3 is t-Bu or Bn. The present invention further relates to a method for preparing liraglutide or a pharmaceutically acceptable salt thereof using the tetrapeptide of formula (I).

Description

Method for preparing tetrapeptide fragment of liraglutide

Cross-reference to related patent applications

This patent application claims priority from indian patent application number 202311008660 filed on day 10, 2, 2023, which is incorporated herein by reference in its entirety.

Technical Field

The present invention relates to an improved process for the preparation of tetrapeptides of formula I for the synthesis of liraglutide,

Wherein R ₁ and R ₂ are independently selected from amine protecting groups and R ₃ is t-Bu or Bn.

The invention further relates to a method for preparing liraglutide or a pharmaceutically acceptable salt thereof using the tetrapeptides of formula I prepared by the method of the invention.

Background

Liraglutide (Liraglutide) is a human GLP-1 receptor agonist (or GLP-1 analog) represented by the formula:

liraglutide (under brand name) (Novo Nordisk) is an antidiabetic agent for the treatment of type 2 diabetes, obesity, and long term weight management. Liraglutide was approved in 2009 in the european union and 2010 in the united states for medical use.

Liraglutide and its preparation are disclosed in US 6268343.

Typically, liraglutide is prepared by chemical synthesis, either by solid phase synthesis or by sequential coupling of amino acids in the liquid phase, or by pooled synthesis involving coupling of separately synthesized fragments.

Most methods known in the art for the synthesis of liraglutide involve the synthesis of a variety of short-chain peptide fragments, such as dipeptides, tripeptides, tetrapeptides, pentapeptides, etc., in solution or solid phase. These fragments are then coupled in the solid phase or in the solution phase or by solid-liquid mixing methods to provide the liraglutide.

The tetrapeptide fragment of formula I is one such fragment useful in the synthesis of liraglutide.

Various methods are known in the art for synthesizing tetrapeptide fragments of formula I having suitable protecting groups in both the solid and liquid phases.

WO2016046753A1 discloses a method for the synthesis of tetrapeptide fragments, wherein the tetrapeptide fragments are prepared by solid phase synthesis, said tetrapeptide fragments having a purity of 94% as determined by HPLC.

CN102875665B and CN106478805B also disclose solid phase synthesis of tetrapeptides of formula I, which are about 94% and 96.6% pure, respectively, as determined by HPLC.

However, solid phase synthesis of short chain peptides, such as tetrapeptides of formula I, is not economically and commercially viable due to the large amounts of solvents used in the process. Recovery of the final product is also cumbersome due to the multiple resin washes, elutions, and purification of the product using large amounts of solvent. There are limitations in the expansion of batch sizes. Therefore, the synthesis of short-chain peptide fragments by solid phase is generally not favored, as this method is not worth the effort involved in the synthesis process.

CN105732798B discloses another method for synthesizing tetrapeptide fragments of formula I. The disclosed method involves the linear coupling of histidine (His) with alanine (Ala), glutamic acid (Glu) and glycine (Gly) in the presence of a solvent and a base.

The tetrapeptides of formula I contain histidine at the amino terminus. Histidine is an amino acid that is extremely racemized. Upon exposure to alkali, the propensity for racemization of histidine increases.

The method as disclosed in CN105732798B results in exposure of histidine amino acids to bases in both stages of synthesis, which leads to racemization of histidine and formation of histidine D-isomer impurities in the tetrapeptides, which impurities are further carried into the final API stage when tetrapeptides with high D-isomer histidine impurities are converted to liraglutide. Such impurities are extremely difficult to separate from the final peptide, and thus the final peptide may contain varying amounts of D-His impurities. The thus obtained liraglutide product has a high level of D-isomer impurities, so that it is unsuitable for use in pharmaceutical formulations.

As is apparent from the above, the reported methods for preparing tetra-peptide fragments of liraglutide require complex operating conditions and do not provide products of high purity, in particular with respect to the D-isomer histidine impurity.

The various methods disclosed in the prior art also involve cumbersome work procedures and multiple purification steps, nor are they cost-effective.

There is therefore still a need to formulate a highly efficient, simple and industrially viable synthesis method which overcomes the drawbacks of the prior art and provides tetrapeptide fragments of formula I as well as liraglutide in a cost-effective manner in high purity.

Object of the Invention

It is an object of the present invention to overcome the above-mentioned drawbacks of the prior art.

It is a further object of the present invention to provide an improved, commercially viable process for the synthesis of tetrapeptide fragments of formula I, and thus of liraglutide using tetrapeptide fragments of formula I prepared by the process described herein.

It is a further object of the present invention to obtain high purity tetrapeptide fragments of formula I and liraglutide or a pharmaceutically acceptable salt thereof.

Disclosure of Invention

The present invention relates to an improved process for the preparation of tetrapeptides of formula I for the synthesis of liraglutide.

In one aspect, the present invention relates to an improved process for the preparation of tetrapeptides of formula I wherein R ₁ and R ₂ are independently selected from amine protecting groups and R ₃ is t-Bu or Bn,

The method comprising the step of condensing an activating compound of formula II with a tripeptide of formula III,

Wherein R ₁ and R ₂ are as defined above and A is an acid activating group,

Wherein R ₃ is as defined above.

In a further aspect, the present invention relates to a process for the preparation of liraglutide or a pharmaceutically acceptable salt thereof, said process comprising converting a tetrapeptide of formula I obtained by the process of the present invention into liraglutide or a pharmaceutically acceptable salt thereof.

Definition of the definition

The following definitions are used in connection with the present application unless the context indicates otherwise.

"Peptide" refers to short chain amino acids in which two or more amino acids are chemically linked by an amide bond.

"Dipeptide" 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.

The term "fragment" refers to a sequence in which two or more amino acids are present. The amino acids in the fragment may be protected or unprotected.

The term "condensation" refers to a condensation reaction that forms an amide bond through the reaction of a carboxyl group with an amine group.

The term "carboxylic acid" refers to an organic compound containing a-COOH group.

The term "amine" refers to an organic compound containing an-NH ₂ group.

The term "activating compound" refers to a compound having an activating ester group.

The term "acid-activating group" refers to a group that enhances the reactivity of a carboxyl group. The activated carboxylic acid reacts the same as its non-activated analogue but faster.

The term "activated ester" refers to an ester functional group that is highly susceptible to nucleophilic attack. The activated ester reacts the same as its unactivated analog, but faster. Activated esters can be prepared by reacting with an activator to convert the hydroxyl (-OH) group of a carboxylic acid group to a good leaving group.

The term "leaving group" refers to an atom, group of atoms or fragment that is detached from the main or remainder of the substrate during the reaction or a substantial step of the reaction.

The term "activator" refers to a compound that reacts with another group or molecule by introducing an activating group into the molecule to activate the molecule or group.

The term "coupling agent" refers to an agent that promotes the formation of a bond between two adjacent groups.

The term "protecting group" refers to a group that is temporarily attached to a functional group to reduce the reactivity of the functional group so that the protected functional group does not react under the synthesis conditions of one or more subsequent steps the molecule undergoes while allowing removal of the protecting group without jeopardizing the remaining molecule.

The term "amine protecting group" refers specifically to a protecting group attached to an amine functional group in a molecule.

For the purposes of the present invention, in molecules having two or more amine protecting groups, the "amine protecting groups" may be the same or different from each other.

The protecting group may be cleaved from the molecule after the desired compound is obtained. The term "deprotection" refers to the cleavage or removal of a protecting group.

The protecting groups may be deprotected under acidic, basic and/or neutral conditions. Methods of protection and deprotection are known in the art (see inter alia "Protective groups in organic synthesis", greene t.w. and Wuts p.g.m., wiley-Interscience, 1999).

The term "ambient temperature" refers to a temperature in the range of about 15 ℃ to 35 ℃.

Detailed Description

The invention relates to a method for producing tetrapeptides of formula I,

In some embodiments, the amine protecting group is selected from the group consisting of t-butoxycarbonyl, trityl, 4-methyltrityl, monomethoxytrityl, fluorenylmethoxycarbonyl, carboxybenzyl, N-benzyloxymethyl, and tosyl.

In one embodiment, R ₁ and R ₂ are the same amine protecting group.

In another embodiment, R ₁ and R ₂ are different amine protecting groups.

In a preferred embodiment, R ₁ is selected from t-butoxycarbonyl, trityl, 4-methyltrityl, monomethoxytrityl, carboxybenzyl, fluorenylmethoxycarbonyl.

In another preferred embodiment, R ₂ is selected from the group consisting of trityl, 4-methyltrityl, monomethoxytrityl, N-benzyloxymethyl, fluorenylmethoxycarbonyl, tosyl and t-butoxycarbonyl.

In a more preferred embodiment, R ₁ is t-butoxycarbonyl and R ₂ is selected from trityl and 4-methyltrityl.

In a still more preferred embodiment, R ₁ is t-butoxycarbonyl and R ₂ is trityl.

In one aspect, the method comprises the step of condensing an activating compound of formula II with a tripeptide of formula III,

Wherein R ₁ and R ₂ are as defined above, A is an acid activating group,

Wherein R ₃ is as defined above.

In one embodiment, the acid activating group A in the compound of formula II is an ester group introduced from an activator selected from the group consisting of N-hydroxysuccinimide, N-hydroxy-5-norbornene-2, 3-dicarboximide, 1-hydroxybenzotriazole, 6-chloro-1-hydroxybenzotriazole, 1-hydroxy-7-azabenzotriazole, 3-hydroxy-4-oxo-3, 4-dihydro-1, 2, 3-benzotriazine, 1-hydroxy-1H-1, 2, 3-triazole-4-carboxylic acid ethyl ester, and N-hydroxytetrazole.

In one embodiment, the condensation reaction of the compound of formula II with the compound of formula III is carried out in the presence of a base and a solvent.

The base may be selected from the group consisting of N, N-diisopropylethylamine, triethylamine, methylmorpholine, sodium bicarbonate, sodium carbonate and potassium carbonate, preferably triethylamine.

The solvent may be selected from the group consisting of dichloromethane, 1-methyl-pyrrolidin-2-one, acetonitrile, tetrahydrofuran, dimethyl sulfoxide, N-dimethylformamide, N-methylformamide, dimethyl carbonate, diethyl carbonate and mixtures thereof. Preferably, the solvent is dichloromethane.

In yet another embodiment, the condensation reaction is carried out at ambient temperature, preferably at a temperature of 20 ℃ to 35 ℃, more preferably at a temperature of 20 ℃ to 30 ℃.

In a preferred embodiment, the tetrapeptides of formula I are compounds of formula Ia [ formula I wherein R ₁ is Boc, R ₂ is Trt and R ₃ is t-Bu ].

In a preferred embodiment, the invention relates to a process for preparing a compound of formula Ia, which comprises condensing an activating compound of formula IIa [ formula II, wherein R ₁ is Boc, R ₂ is Trt and a is ONB ] with a tripeptide of formula IIIa [ formula III, wherein R ₃ is t-Bu ].

In a further preferred embodiment, the condensation is carried out in the presence of methylene chloride and triethylamine at a temperature of from 25 ℃ to 30 ℃. The reaction mixture was stirred for 2-4 hours and the tetrapeptides of formula Ia were isolated from the reaction mixture after extraction with dichloromethane and precipitation with ethyl acetate.

In another embodiment, the activated compound of formula II is prepared by activating the carboxyl group of the compound of formula IV using an activator,

Wherein R ₁ and R ₂ are as defined above.

The activator may be selected from the group consisting of N-hydroxysuccinimide, N-hydroxy-5-norbornene-2, 3-dicarboximide, 1-hydroxybenzotriazole, 6-chloro-1-hydroxybenzotriazole, 1-hydroxy-7-azabenzotriazole, 3-hydroxy-4-oxo-3, 4-dihydro-1, 2, 3-benzotriazine, 1-hydroxy-1H-1, 2, 3-triazole-4-carboxylic acid ethyl ester and N-hydroxytetrazole. Preferably, the activator is N-hydroxy-5-norbornene-2, 3-dicarboximide.

In one embodiment, the activation of the carboxyl group is performed in the presence of a coupling agent in a solvent.

The coupling agent may be selected from the group consisting of N- (3-dimethylaminopropyl) -N ' -ethylcarbodiimide hydrochloride, N, N-dicyclohexylcarbodiimide, oxyma B/diisopropylcarbodiimide, benzotriazol-1-yl-oxy-tri-pyrrolidinyl-phosphonium hexafluorophosphate, azabenzotriazole tetramethylurea, O- (1H-benzotriazol-1-yl) -N, N, N ', N ' -tetramethylurea hexafluorophosphate, O-benzotriazole-N, N, N ', N ' -tetramethylurea tetrafluoroborate, and propylphosphoric anhydride. Preferably, the coupling agent is selected from the group consisting of N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride, N-dicyclohexylcarbodiimide and Oxyma B/diisopropylcarbodiimide. More preferably, the coupling agent is N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride.

The solvent may be selected from the group consisting of dichloromethane, 1-methyl-pyrrolidin-2-one, acetonitrile, tetrahydrofuran, dimethyl sulfoxide, N-dimethylformamide, N-methylformamide, dimethyl carbonate, diethyl carbonate and mixtures thereof. Preferably, the activation of the carboxyl groups using the activator is performed in tetrahydrofuran in the presence of a coupling agent.

In a preferred embodiment, the compound of formula IV (wherein R ₁ is Boc and R ₂ is Trt) is activated using N-hydroxy-5-norbornene-2, 3-dicarboximide (henb) to form an activated compound of formula IIa [ formula II wherein R ₁ is Boc, R ₂ is Trt and a is ONB ].

In another preferred embodiment, the compound of formula IV is treated with N-hydroxy-5-norbornene-2, 3-dicarboximide (henb) in the presence of N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride in tetrahydrofuran at a temperature of 20-30 ℃ for 10 to 12 hours. After completion of the reaction, the activated compound of formula II is isolated by adding water and dichloromethane.

In one embodiment, the present invention relates to a process for the preparation of a tripeptide of formula III, comprising the step of activating the carboxyl group of a compound of formula V in a solvent with an activating agent in the presence of a coupling agent to obtain an activated compound of formula VI,

Wherein R ₄ is an amine protecting group,

Wherein R ₄ and A are as defined above.

The amine protecting group (R ₄) may be selected from fluorenylmethoxycarbonyl, t-butoxycarbonyl, carboxybenzyl and tosyl, preferably it is fluorenylmethoxycarbonyl.

In a preferred embodiment, the compound of formula V, wherein R ₄ is Fmoc, is activated using N-hydroxy-5-norbornene-2, 3-dicarboximide (HONB) to yield an activated compound of formula VI, wherein R ₄ is Fmoc; A is ONB.

In another preferred embodiment, the compound of formula V is treated with N-hydroxy-5-norbornene-2, 3-dicarboximide (henb) in the presence of N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride at 25 ℃ to 30 ℃ in tetrahydrofuran for 10 to 12 hours to yield the activated compound of formula VI. After completion of the reaction, the activated compound of formula VI is isolated in dichloromethane by adding water and dichloromethane.

In one embodiment, the activated compound of formula VI is further condensed with the dipeptide of formula VII in a solvent in the presence of a base to obtain a protected tripeptide of formula VIII,

Wherein R ₃ is t-Bu or Bn,

Wherein R ₃ is t-Bu or Bn and R ₄ is an amine protecting group.

The base may be selected from the group consisting of N, N-diisopropylethylamine, triethylamine, methylmorpholine, sodium bicarbonate, sodium carbonate and potassium carbonate. Preferably the base is triethylamine.

In a preferred embodiment, the activating compound of formula VI (wherein R ₄ is Fmoc; A is ONB) is condensed with the dipeptide of formula VII (wherein R ₃ is t-Bu) to provide a protected tripeptide of formula VIII (wherein R ₃ is t-Bu and R ₄ is Fmoc).

In another preferred embodiment, the condensation is carried out in the presence of triethylamine and with methylene chloride as solvent.

Deprotection of the protected tripeptide of formula VIII to obtain the tripeptide of formula III is accomplished by any method known in the art. Deprotection is carried out in acid or base, depending on the protecting group to be removed.

Preferably, the protected tripeptide of formula VIII is deprotected in a solvent in the presence of an organic base.

The organic base may 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. Preferably, the organic base is diethylamine.

The solvent in the deprotection reaction may be selected from the group consisting of dichloromethane, 1-methyl-pyrrolidin-2-one, acetonitrile, tetrahydrofuran, dimethyl sulfoxide, N-dimethylformamide, N-methylformamide, dimethyl carbonate, diethyl carbonate and mixtures thereof. Preferably, the solvent is dichloromethane.

More preferably, the deprotection of the protected tripeptide of formula VIII may be performed in the presence of diethylamine in a dichloromethane solvent.

Preferably, the deprotection is carried out at a temperature of 30 ℃ to 50 ℃, more preferably at a temperature of 35 ℃ to 45 ℃. The solids may be separated from the reaction mixture by any suitable method. In certain embodiments, the solids are isolated by adding diisopropyl ether, filtration, and drying.

The dipeptide of formula VII is obtained by a process comprising the steps of activating the carboxyl group of a compound of formula IX with an activator to provide a compound of formula X,

Wherein R ₅ is an amine protecting group,

Wherein R ₅ and A are as defined above.

The amine protecting group R ₅ may be selected from the group consisting of fluorenylmethoxycarbonyl, t-butoxycarbonyl, carboxybenzyl and tosyl, preferably it is fluorenylmethoxycarbonyl.

The activation of formula IX is carried out by an activator in the presence of a coupling agent and a solvent.

In a preferred embodiment, the compound of formula IX, wherein R ₅ is Fmoc, is activated with N-hydroxy-5-norbornene-2, 3-dicarboximide (HONB) to yield an activated compound of formula X, wherein R ₅ is Fmoc; A is ONB.

In a preferred embodiment, the compound of formula IX is treated with N-hydroxy-5-norbornene-2, 3-dicarboximide (henb) in the presence of N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride in tetrahydrofuran at 20-30 ℃ for 6 to 8 hours, resulting in the activation of the compound of formula IX. After the reaction is completed, the obtained compound of formula X is extracted in dichloromethane by adding water and dichloromethane.

The starting compound of formula IX (wherein R ₅ is Fmoc) is available from commercial sources.

Subsequently, the activated compound of formula X is condensed with glycine (Gly) to provide a protected dipeptide which upon deprotection yields the dipeptide of formula VII.

In a preferred embodiment, the condensation of the compound of formula X with glycine is carried out in the presence of a solvent, preferably dichloromethane, and a base, preferably triethylamine.

Deprotection of the protected dipeptide obtained as described above to obtain the dipeptide of formula VII is carried out by any method known in the art. Deprotection is carried out in acid or base, depending on the protecting group to be removed.

Preferably, the protected dipeptide is reacted with an organic base in a solvent.

The solvent used for the deprotection reaction may be selected from the group consisting of dichloromethane, 1-methyl-pyrrolidin-2-one, acetonitrile, tetrahydrofuran, dimethyl sulfoxide, N-dimethylformamide, N-methylformamide, dimethyl carbonate, diethyl carbonate and mixtures thereof in the presence of an organic base. Preferably, the solvent is dichloromethane.

More preferably, the deprotection is carried out in the presence of diethylamine in a dichloromethane solvent. Preferably, the deprotection is carried out at 20-30 ℃ for 5 to 8 hours. The solids may be separated from the reaction mixture by any suitable method. In some embodiments, the solid is isolated by adding water and methylene chloride, followed by filtration and drying to produce the dipeptide of formula VII.

In some embodiments, the activated ester compounds of formulas II, VI and X obtained in the process of the present invention are prepared in situ and are not isolated during the process.

In the process of the present application, the tetrapeptides of formula I are prepared by using histidine in the final stage of the reaction sequence. This limits the exposure of histidine to alkali during the process. Using the methods described herein, the exposure of the histidine residues/histidine-containing peptides to the base occurs only once during synthesis, thus racemization of histidine to the D-isomer is minimal.

Surprisingly, the inventors have found that by sequential condensation of amino acids with fragments according to the reaction sequence described in the method of the application, e.g. by introducing histidine in the final stage, the D-isomer impurity of histidine in the tetrapeptide fragments is highly reduced.

In contrast, the prior art methods disclose the introduction of histidine in the first stage, whereby the histidine residues are exposed to base in each stage in the subsequent reaction step until the tetrapeptide formation is complete. This results in racemization of histidine during the process, resulting in a product with high impurity content, in particular high content of D-histidine impurities.

The inventors also tried to synthesize tetrapeptides of formula I by introducing histidine in the second stage instead of the first stage to reduce histidine exposure to alkali during the process. This also resulted in increased racemization of histidine, resulting in about 3% D-His impurity, whereas the method of the present invention produced tetrapeptide fragments with negligible D-isomer content of histidine, without any further purification. The tetrapeptides of formula I obtained by the process of the present invention contain less than 0.5% of histidine D-isomer impurities as determined by HPLC, preferably less than 0.4% of histidine D-isomer impurities as determined by HPLC, more preferably less than 0.3% of histidine D-isomer impurities as determined by HPLC, even more preferably less than 0.2% of histidine D-isomer impurities as determined by HPLC.

The process described in the present invention results in a tetrapeptide of formula I having a very low content of histidine D-isomer impurities (e.g. less than 0.5% as determined by HPLC) even in the crude tetrapeptide. In addition, the crude product may be purified using methods known in the art to remove unreacted compounds (e.g., amino acids). However, according to the present invention, the purification of the crude tetrapeptide is only used to remove unreacted starting amino acids, and not necessarily to remove D-His impurities, which have been controlled in the crude product obtained by using the method of the present invention.

In some embodiments, the solvent used to purify the tetrapeptides of formula I is selected from the group consisting of ethyl acetate, acetone, isopropanol, tetrahydrofuran, acetonitrile, and mixtures thereof.

The tetrapeptides of formula I obtained by the process of the present invention have a purity of 98% or more as determined by HPLC, preferably 99% or more as determined by HPLC, most preferably 99.4% as determined by HPLC.

Preferably, the process according to the invention is carried out in the solution phase. This also eliminates the drawbacks of solid phase synthesis such as the use of expensive resins, multiple washes with solvents, and large scale batch size.

In a further embodiment, the tetrapeptides of formula I obtained by the methods of the invention may be converted to liraglutide or a pharmaceutically acceptable salt thereof by methods known in the art, for example using the methods reported in WO2016046753 A1.

The use of the high purity tetrapeptide fragments of formula I obtained by the method of the present invention positively affects the yield and purity of liraglutide or a salt thereof.

Accordingly, the present invention provides liraglutide or a pharmaceutically acceptable salt thereof, which contains less than 0.5% histidine D-isomer impurity as determined by HPLC, preferably less than 0.4% histidine D-isomer impurity as determined by HPLC, more preferably less than 0.3% histidine D-isomer impurity as determined by HPLC, even more preferably less than 0.2% histidine D-isomer impurity as determined by HPLC.

Accordingly, the inventors of the present invention developed an improved method for synthesizing not only tetrapeptide fragments, but also liraglutide or a pharmaceutically acceptable salt thereof, which is both cost-effective and commercially viable.

The process can be scaled up efficiently and the products of the various stages of the synthesis or their intermediates can be separated using separation techniques such as solvent extraction and solvent recovery, precipitation, distillation, filtration and product drying.

Therefore, the synthesis method of the invention improves the purity of the peptide, reduces the raw material and purification cost, and is beneficial to industrial production.

Abbreviations/abbreviations:

HONB N-hydroxy-5-norbornene-2, 3-dicarboximide, and accordingly ONB refers to the same molecule when it is attached to a second molecule via a hydroxy group (so that there is no H on the hydroxy group).

EDC. HCl N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride

DCC dicyclohexylcarbodiimide

Boc

Fmoc fluorenylmethoxycarbonyl

Trt trityl radical

Bn-: benzyl-)

His-histidine

Ala-Ala

Glu, glu

Gly-glycine

T-Bu t-butyl

OH hydroxyl group

D-isomer, D-isomer

HPLC high performance liquid chromatography

Detailed Description

Experiment

The detailed experimental parameters described in the present invention are provided by the following examples which are intended to be illustrative and not limiting of all possible embodiments of the invention.

Examples:

Comparative examples

Preparation of tetrapeptides (Boc) -His (Trt) -Ala-Glu (OtBu) -Gly-OH

Stage-1 preparation of dipeptide Glu (OtBu) -Gly-OH

A) EDC. HCl (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 ℃ for 15-16 hours. Distilled water (750 ml) and a brine solution (150 ml) were added to the reaction mixture, and stirred for 190 minutes, after which standing was allowed. The aqueous layer was separated and discarded. Water (750 ml) and brine solution (150 ml) were added to the organic layer. The mixture was stirred. The layers were separated and the aqueous layer was discarded.

B) Triethylamine solution (35.7 g in 150ml of tetrahydrofuran) and glycine solution (26.5 g in 300ml of distilled water) were slowly added to the organic layer at 20-30℃and the reaction mixture was stirred for 3 to 5 hours. Distilled water (600 ml) and ethyl acetate (600 ml) were added to the reaction mixture, and the pH was adjusted to 3 to 4. The reaction was stirred at 20-30 ℃ for 10 minutes. Allow to stand and separate the layers. Sodium bicarbonate solution (52.5 g in 750ml distilled water) and brine solution (150 ml) were added to the organic layer, and the mixture was stirred at 20-30 ℃ for 15 minutes, after which time the layers were left to stand and separated. Distilled water was added to the organic layer, and the pH of the reaction was adjusted to 3 to 4. The reaction was stirred for 10 minutes, after which time it was allowed to stand and the layers separated. The aqueous layer was discarded and the organic layer was recovered under vacuum at a temperature below 40 ℃. The residue was degassed under vacuum for 120 min, and methylene chloride (300 ml) was added to the residue. The mixture was stirred to give a clear solution and heated to 35 to 40 ℃. The solid was isolated from the reaction mixture by addition of diisopropyl ether, cooling, filtration and drying under vacuum.

C) Dichloromethane (450 ml) and Fmoc-Glu (OtBu) -Gly (90 gm) were stirred at 20-30℃for 5-10 min. Diethylamine (90 ml) was added and the resulting mixture was stirred at 20-30 ℃ for 5-7 hours. Distilled water (360 ml) was added to the reaction and stirred for 10 minutes. The mixture was allowed to stand and the layers were separated. Dichloromethane (450 ml) was added to the aqueous layer and stirred for 10-15 minutes. The organic layer was discarded and the aqueous layer was distilled under vacuum at a temperature below 55 ℃. Methylene chloride (300 ml) was added to the residue, and the residue was recovered under vacuum at a temperature below 45 ℃. The residue was degassed under vacuum at 35-45 ℃ for 1-2 hours. The solid (44.5 g) was isolated by adding dichloromethane, then filtering, washing with dichloromethane and drying under vacuum at a temperature of 40-50 ℃ for 14-16 hours.

Yield 48.5% (purity: 98.2%).

Stage-2 preparation of dipeptide Boc-His (Trt) -Ala-OH

EDC. HCl (38.6 g) was added to a mixture of HONB (36 g) and Boc-His (Trt) -OH (50 g) in acetonitrile (225 ml). The mixture was stirred overnight at 20-30 ℃ until the reaction was complete. The reaction was cooled to 0-10 ℃ and L-alanine solution (9 g in 125ml deionized water) and triethylamine solution (20.4 gm in 50ml acetonitrile) were added to the reaction. Distilled water (1000 ml) and ethyl acetate (500 ml) were added to the reaction. The pH was adjusted to 3.9 with hydrochloric acid and the layers were separated. The aqueous layer was discarded. The organic layer was washed with sodium bicarbonate solution (70 gm in 1000ml 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, then distilled at 40-50 ℃ and degassed. Acetonitrile (500 ml) was added to the resulting mass and heated to 40-50 ℃ for 30 minutes, then cooled to 35-40 ℃. Dichloromethane (50 ml) was added to the reaction mixture and stirred for 1 hour, then cooled to 20-25 ℃. The reaction mixture was stirred at 20-30 ℃ for 4 hours, after which time the solid was filtered and washed with acetonitrile (100 ml) followed by n-hexane (100 ml). The reaction was filtered and dried in vacuo to give a solid (42 g).

The yield was 73.4%.

Stage-3 preparation of tetrapeptides (Boc) -His (Trt) -Ala-Glu (OtBu) -Gly-OH)

DCC (10.86 g) was added to a mixture of HONB (3.04 g) and (Boc) -His (Trt) -Ala-OH (10 g) in tetrahydrofuran (125 ml). The mixture was stirred overnight at 20-30 ℃ until the reaction was complete. The reaction was cooled to 0-5 ℃, filtered and washed with tetrahydrofuran (20 ml). Glu (OtBu) -Gly-OH (5.5 gm) in tetrahydrofuran (10 ml) was added to the reaction at 0-10 ℃. Triethylamine (5 ml) was added and the reaction stirred at 20-30℃for 2 hours until completion. The reaction was evaporated under vacuum at 35-40 ℃ and the residue obtained was diluted with ethyl acetate (150 ml). Water was added to the dilution and the pH was adjusted to 3.4-3.5 with hydrochloric acid solution. The organic layer was separated and washed with saturated NaHCO ₃ solution (100 ml). Water was added to the organic layer and 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 ℃. Water and methylene chloride (50 ml) were added to the reaction and stirred. The dichloromethane layer was separated and slowly added to diisopropyl ether (150 ml). The precipitate was filtered, washed with diisopropyl ether and dried under vacuum to give a solid (6 g).

Yield 42.07% (purity: 85.1%) and D-isomer 2.99%.

Example-1

Preparation of tetrapeptides (Boc) -His (Trt) -Ala-Glu (OtBu) -Gly-OH

Stage-1 preparation of dipeptide Glu (OtBu) -Gly-OH

A) EDC. HCl (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 ℃ for 15-16 hours. Distilled water (750 ml) and a brine solution (150 ml) were added to the reaction mixture, and stirred for 190 minutes, after which it was allowed to stand. The aqueous layer was separated and discarded. Water (750 ml) and brine solution (150 ml) were added to the organic layer. The mixture was stirred. The layers were separated and the aqueous layer was discarded.

C) Dichloromethane (450 ml) and Fmoc-Glu (OtBu) -Gly (90 gm) were stirred at 20-30℃for 5-10 min. Diethylamine (90 ml) was added and the resulting mixture was stirred at 20-30 ℃ for 5-7 hours. Distilled water (360 ml) was added to the reaction mixture and stirred for 10 minutes. The mixture was allowed to stand and the layers were separated. Dichloromethane (450 ml) was added to the aqueous layer and stirred for 10-15 minutes. The layers were separated again. The aqueous layer was distilled under vacuum at a temperature below 55 ℃. Methylene chloride (300 ml) was added to the residue and recovered under vacuum at a temperature below 45 ℃. The residue was degassed at 35-45 ℃ under vacuum for 1 to 2 hours. The solid (44.5 g) was isolated by adding dichloromethane, then filtering, washing with dichloromethane and drying under vacuum at a temperature of 40-50 ℃ for 14-16 hours.

Yield 48.5% (purity: 98.2%).

Stage-2 preparation of dipeptide Ala-Glu (OtBu) -Gly-OH

A) EDC. HCl (92.40 g) was added to a mixture of HONB (86.32 g) and Fmoc-Ala (100 g) in tetrahydrofuran (500 ml) and stirred at 20-30℃for 10 to 12 hours. After the completion of the reaction, distilled water and methylene chloride (500 ml) were added to the reaction mixture and stirred for 10 to 15 minutes, followed by allowing to stand. The aqueous layer was separated and the product extracted with dichloromethane solvent. The dichloromethane layers were combined and water was added. The layers were separated and the aqueous layer was discarded. The organic layer is preserved.

B) Glu (OtBu) -Gly-OH (83.59 g) was added to the dichloromethane layer at 20-30 ℃. Triethylamine (48.75 g) and water were added to the mixture, and the mixture was stirred for 2 hours. The solvent was evaporated under vacuum at 40 to 45 ℃. Methanol and distilled water were added to the residue and the reaction mixture was cooled to 20-30 ℃. The pH of the reaction mixture was adjusted to 3.0 to 4.0 and stirred for 2 to 4 hours. The solid was isolated from the reaction mixture by filtration, washing with water and extraction with methanol at a temperature of 35 to 45 ℃ and cooling to 20-30 ℃.

C) Dichloromethane (500 ml) and diethylamine (100 ml) were added to the solid obtained in step b) (Fmoc-Ala-Glu (OtBu) -Gly-OH) at 20-30 ℃. The reaction was stirred for 5-8 hours and distilled water was added. The layers were separated and the aqueous layer was recovered at less than 50 ℃ under vacuum. Dichloromethane (500 ml) was added to the above residue and the mixture was heated to 35-45 ℃. The solid (71 g) was isolated from the reaction mixture by addition of diisopropyl ether, filtration and drying at 40-50 ℃ under vacuum for 15-18 hours.

Yield 66.7% (purity: 94.8%).

Stage-3 preparation of tetrapeptides (Boc) -His (Trt) -Ala-Glu (OtBu) -Gly-OH)

A) EDC. HCl (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 ℃ for 10 to 12 hours. After the completion of the reaction, distilled water and methylene chloride (500 ml) were added to the reaction mixture, and stirred for 10 to 15 minutes, followed by allowing to stand.

B) Ala-Glu (OtBu) -Gly-OH (66.57 g) was added to the dichloromethane layer from the above step, and triethylamine (30.5 g) and distilled water (50 ml) were added thereto. The reaction was stirred at 20-30 ℃ for 2 to 4 hours and the pH of the reaction was adjusted to 3.0 to 4.0. The layers were separated. The aqueous layer was further washed with dichloromethane. The dichloromethane layers were combined and saturated sodium bicarbonate solution (500 ml) was added thereto and stirred for 10-15 minutes. The reaction mixture was allowed to stand and the layers were separated. The organic layer was recovered under vacuum at a temperature below 45 ℃ to obtain a residue (crude; D-isomer: 0.34%).

Ethyl acetate (500 ml) was added to the residue and the temperature was raised to 35-45 ℃. The reaction was stirred at 35-45 ℃ for 2-3 hours and cooled to a temperature of 20-30 ℃. The solid (118 g) was isolated by filtration and washing with ethyl acetate and drying at 40-45 ℃ under vacuum for 15-18 hours.

Yield 72.4% (purity 99.4%), D-isomer 0.22%.

Example-2

Preparation of tetrapeptides (Boc) -His (Trt) -Ala-Glu (OtBu) -Gly-OH

Stage-1 preparation of dipeptide Glu (OtBu) -Gly-OH

A) EDC. The mixture was stirred at 20-30 ℃ for 12-15 hours. The reaction mixture was cooled to 10-15 ℃. Pre-chilled distilled water (1000 ml) and cold brine solution were added to the reaction mixture and stirred. Allow to stand, separate the layers, and discard the aqueous layer. The organic layer was washed with pre-chilled distilled water (1000 ml) and cold brine solution. The mixture was again allowed to stand and the aqueous layer was discarded.

B) Glycine solution and triethylamine solution were added to the organic layer at 10-20 ℃. The reaction mixture was stirred for 4-6 hours, after which the reaction mixture was cooled to 10-15 ℃. Precooled distilled water (800 ml) and precooled ethyl acetate (2000 ml) were added thereto at 10-15 ℃. The pH was adjusted to 3 to 3.5. The reaction mixture was stirred at 10-15 ℃ for 10-15 minutes after which the layers were allowed to stand and the aqueous layer was discarded. Pre-chilled sodium bicarbonate solution and cold brine solution were added to the reaction mixture (organic layer) at 10-20 ℃. The reaction mixture was stirred and the layers were separated. Precooled distilled water (1000 ml) was added to the organic layer at 10-15 ℃. The pH was adjusted to 3 to 3.5 and the reaction mixture was stirred for 10-15 minutes. The layers were allowed to stand and separated, and the aqueous layer was discarded. The organic layer was recovered under vacuum at a temperature below 50 ℃. Dichloromethane (400 ml) was added and then recovered under vacuum at a temperature below 50 ℃. The residue was degassed under vacuum and dichloromethane (400 ml) was added thereto. Stirring to give a clear solution. The reaction was heated to 35-40 ℃ and diisopropyl ether (2000 ml) was added and the reaction mixture was stirred at 35-45 ℃ for 30-60 minutes. The reaction mixture was cooled to 20-30 ℃ and stirred for 2-3 hours, and the obtained solid was filtered. The solid was washed with diisopropyl ether (200 ml) and dried under vacuum for 100-120 minutes for the next step as such.

Dichloromethane (400 ml) was added to the residue and stirred to give a clear solution. The reaction mixture was heated to 35-40 ℃ and diisopropyl ether (200 ml) was added thereto. The mixture was stirred at 35-45 ℃ for 30-60 minutes and cooled to 20-30 ℃. The mixture was stirred for 2-3 hours and the solid obtained was filtered, washed with diisopropyl ether and dried under vacuum.

C) Dichloromethane (1000 ml) was added to the residual solid and the reaction mixture was stirred at 20-30 ℃ for 5-10 minutes. Diethylamine (200 ml) was added thereto and the mixture was stirred for 8-10 hours. Distilled water (1000 ml) was added to the reaction mixture and stirred for 20-25 minutes. The layers were separated. The aqueous layer was washed with dichloromethane and distilled under vacuum at a temperature below 50 ℃. Dichloromethane (400 ml) was added thereto and then recovered under vacuum at a temperature below 40 ℃. Dichloromethane (1000 ml) was added to the residue, heated to 30-40 ℃, and diisopropyl ether (1000 ml) was then added to the reaction at 30-40 ℃ and stirred for 60 minutes. The reaction mixture was cooled to 20 to 30 ℃ and stirred for 2-3 hours. The solid (74 g) was isolated by filtration, washed with dichloromethane and dried under vacuum.

Yield 60.6% (purity 99.87%)

Stage-2 preparation of tripeptide Ala-Glu (OtBu) -Gly-OH

A) EDC. HCl (74.03 g) was added to a mixture of HONB (69.16 g) and Fmoc-Ala (80 g) in tetrahydrofuran (640 ml) and stirred at 25 to 30℃for 12-15 hours. The reaction mixture was cooled to 10-15 ℃. Pre-chilled distilled water (400 ml) and cold brine solution were added to the reaction mixture and stirred.

Allow to stand the layers and discard the aqueous layer. The organic layer was washed with pre-chilled distilled water (400 ml) and cold brine solution, the mixture was allowed to stand and the aqueous layer was discarded again.

B) Glu (OtBu) -Gly-OH (66.89 g), triethylamine (39.06 g) and water were added to the organic layer at 10-20℃and the mixture was stirred at 25-30℃for 4-6 hours. The solvent was removed under vacuum at 40-45 ℃ and the reaction mixture was degassed and then cooled to 10-15 ℃. Pre-chilled methanol (400 ml) and chilled water (800 ml) were added to the residue and the temperature was adjusted to 10-20 ℃. The pH of the reaction mixture was slowly adjusted to 3.0 to 3.5. The reaction mixture was stirred for 2-4 hours and the solid obtained was filtered, washed with cold water and dried under vacuum for 1-2 hours.

Methanol (400 ml) was added to the obtained solid (Fmoc-Ala-Glu (OtBu) -Gly-OH) at 20-30℃and the temperature was raised to 40-50℃after which the reaction mixture was stirred for 1-2 hours. The reaction mixture was cooled to 20-30 ℃ and stirred for 1-2 hours, after which the obtained solid was filtered. The solid was washed with methanol and dried under vacuum.

Methanol (400 ml) was added to the solid obtained above, and the temperature of the reaction mixture was raised to 40-50 ℃, then cooled to 20-30 ℃ and stirred for 1-2 hours. The solid obtained is filtered, washed with methanol and dried under vacuum for 1-2 hours.

C) Dichloromethane (400 ml) and diethylamine (80 ml) were added to the solid at 20-30 ℃ and the reaction mixture was stirred for 7-9 hours. Distilled water (400 ml) was added to the reaction mixture and stirred for 20 to 25 minutes, followed by standing. The layers were separated. The aqueous layer was washed with dichloromethane and then distilled under vacuum at a temperature below 50 ℃. Dichloromethane (400 ml) was added thereto and then recovered under vacuum at a temperature below 40 ℃. Dichloromethane (400 ml) was again added and the residue heated to 30-40 ℃. Diisopropyl ether (400 ml) was added and the reaction stirred for 60 minutes and cooled to 20-30 ℃ and stirred for a further 2-3 hours. The solid (46 g) was isolated by filtration, washed with diisopropyl ether and dried under vacuum.

Yield 50.94% (purity 98.3%).

Stage-3 preparation of tetrapeptides (Boc) -His (Trt) -Ala-Glu (OtBu) -Gly-OH)

A) EDC. HCl (34.52 g) was added to a mixture of HONB (32.25 g) and Boc-His (Trt) -OH (60 g) in tetrahydrofuran (480 ml). The mixture was stirred at 25-30 ℃ for 12-15 hours and cooled to 10-15 ℃. Pre-chilled distilled water (300 ml) and cold brine solution were added simultaneously to the reaction mixture and stirred. The layers were allowed to stand and separated, and the aqueous layer was discarded.

The organic layer was washed with pre-chilled distilled water (300 ml) and cold brine solution. The layers were separated.

B) Ala-Glu (OtBu) Gly-OH (39.76 g) was added to the organic layer, and triethylamine (18.21 g) and distilled water (60 ml) were added thereto. The reaction mixture was stirred at 25-30 ℃ for 2-4 hours and then cooled to 10-15 ℃. Precooled distilled water (300 ml) and precooled dichloromethane (600 ml) were added thereto, and the pH was adjusted to 3 to 3.5, and the mixture was stirred and allowed to stand to separate the layers. The organic layer was washed with a pre-chilled sodium bicarbonate solution and a pre-chilled brine solution at 10-15 ℃. The aqueous layer was discarded.

Precooled distilled water (300 ml) was added to the organic layer and the pH of the reaction mixture was adjusted to 3 to 3.5. The mixture was stirred and allowed to stand to separate the layers. The organic layer was recovered under vacuum and degassed. Ethyl acetate (600 ml) was added to the obtained residue and the temperature was raised to 40-45 ℃, after which the material was cooled to 20-30 ℃, stirred, filtered to obtain a solid. The solid was washed with ethyl acetate and dried under vacuum.

Methylene chloride (2400 ml) was added to the solid and the reaction mixture was heated to 33-35 ℃ and stirred to give a clear solution. Distilled water (600 ml) and a brine solution were added thereto, and the reaction mixture was stirred, and left to stand to separate the layers.

The organic layer was washed with distilled water and brine solution, recovered at a temperature below 50 ℃ under vacuum and degassed to obtain a residue.

Ethyl acetate (600 ml) was added to the residue. The temperature was raised to 40-45 ℃. The reaction was cooled to 20-30 ℃, stirred, and filtered to obtain a solid. The solid (63 g) was washed with ethyl acetate (60 ml) and dried under vacuum.

Yield 64.81% (purity: 98.4%), D-isomer 0.04%.

Claims

1. A method for preparing a tetrapeptide of formula I,

The method comprises the steps of condensing an activated compound of formula II and a tripeptide of formula III,

wherein R ₁ and R ₂ are independently selected from amine protecting groups, R ₃ is t-Bu or Bn, and A is an acid activating group.

2. The method according to claim 1, wherein R ₁ is selected from tert-butyloxycarbonyl, trityl, 4-methyltrityl, monomethoxytrityl, carboxybenzyl, fluorenylmethoxycarbonyl; and R ₂ is selected from trityl, 4-methyltrityl, monomethoxytrityl, N-benzyloxymethyl, fluorenylmethoxycarbonyl, tosyl and tert-butyloxycarbonyl.

3. The method according to claim 1, wherein the acid-activated group A is an ester group introduced from an activator selected from the group consisting of N-hydroxysuccinimide, N-hydroxy-5-norbornene-2,3-dicarboximide, 1-hydroxybenzotriazole, 6-chloro-1-hydroxybenzotriazole, 1-hydroxy-7-azabenzotriazole, 3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine, 1-hydroxy-1H-1,2,3-triazole-4-carboxylic acid ethyl ester and N-hydroxytetrazole.

The method according to claim 1 , wherein the condensation reaction is carried out in the presence of a base.

5 . The method according to claim 4 , wherein the base is selected from the group consisting of N,N-diisopropylethylamine, triethylamine, methylmorpholine, sodium bicarbonate, sodium carbonate and potassium carbonate.

The method according to claim 1 , wherein the condensation reaction is carried out in the presence of a solvent.

7. The method according to claim 6, wherein the solvent is selected from the group consisting of dichloromethane, 1-methyl-pyrrolidin-2-one, acetonitrile, tetrahydrofuran, dimethyl sulfoxide, N,N-dimethylformamide, N-methylformamide, dimethyl carbonate, diethyl carbonate, and mixtures thereof.

8. The process of claim 1, wherein the condensation reaction is carried out at ambient temperature.

9. The method according to claim 1, wherein the compound of formula II is prepared by activating the carboxyl group of the compound of formula IV using an activating agent in a solvent in the presence of a coupling agent.

10. The method according to claim 9, wherein:

The activator is selected from the group consisting of: N-hydroxysuccinimide, N-hydroxy-5-norbornene-2,3-dicarboximide, 1-hydroxybenzotriazole, 6-chloro-1-hydroxybenzotriazole, 1-hydroxy-7-azabenzotriazole, 3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine, 1-hydroxy-1H-1,2,3-triazole-4-carboxylic acid ethyl ester and N-hydroxytetrazole,

The coupling agent is selected from the group consisting of: N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, N,N-dicyclohexylcarbodiimide, Oxyma B/diisopropylcarbodiimide, benzotriazol-1-yl-oxy-tris-pyrrolidinyl-phosphonium hexafluorophosphate, azabenzotriazole tetramethyluronium hexafluorophosphate, O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate and propylphosphonic anhydride, and

The solvent is selected from the group consisting of dichloromethane, 1-methyl-pyrrolidin-2-one, acetonitrile, tetrahydrofuran, dimethyl sulfoxide, N,N-dimethylformamide, N-methylformamide, dimethyl carbonate, diethyl carbonate, and mixtures thereof.

11. The method of claim 1 , wherein the tripeptide of formula III is prepared by a method comprising the following steps:

a) activating the carboxyl group of the compound of formula V using an activating agent in a solvent in the presence of a coupling agent to obtain an activated compound of formula VI, wherein R ₄ is an amine protecting group,

b) condensing an activated compound of formula VI with a dipeptide of formula VII in a solvent in the presence of a base to obtain a protected tripeptide of formula VIII,

c) deprotecting the protected tripeptide of formula VIII in a solvent in the presence of an organic base.

12. The method according to claim 11, wherein _R4 is selected from fluorenylmethyloxycarbonyl, tert-butyloxycarbonyl, carboxybenzyl and toluenesulfonyl.

13. The method according to claim 11, wherein:

The activator in step a) is selected from the group consisting of: N-hydroxysuccinimide, N-hydroxy-5-norbornene-2,3-dicarboximide, 1-hydroxybenzotriazole, 6-chloro-1-hydroxybenzotriazole, 1-hydroxy-7-azabenzotriazole, 3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine, 1-hydroxy-1H-1,2,3-triazole-4-carboxylic acid ethyl ester and N-hydroxytetrazole;

The coupling agent in step a) is selected from the group consisting of: N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, N,N-dicyclohexylcarbodiimide, Oxyma B/diisopropylcarbodiimide, benzotriazol-1-yl-oxy-tris-pyrrolidinyl-phosphonium hexafluorophosphate, azabenzotriazole tetramethyluronium hexafluorophosphate, O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate and propylphosphonic anhydride;

The base in step b) is selected from the group consisting of: N,N-diisopropylethylamine, triethylamine, methylmorpholine, sodium bicarbonate, sodium carbonate and potassium carbonate;

The organic base in step c) is selected from the group consisting of ammonia, piperidine, piperazine, tributylamine, diethylamine, pyrrolidine, ethanolamine, morpholine, 1,8-diazabicyclo[5.4.0]undec-7-ene, 1,4-diazabicyclo[2.2.2]octane and dicyclohexylamine; and

The solvent in step a), b) or c) is selected from the group consisting of dichloromethane, 1-methyl-pyrrolidin-2-one, acetonitrile, tetrahydrofuran, dimethyl sulfoxide, N,N-dimethylformamide, N-methylformamide, dimethyl carbonate, diethyl carbonate and mixtures thereof.

14. The method of claim 1, wherein the tetrapeptide of formula I is a tetrapeptide of formula Ia

15. The method according to claim 14, wherein the tetrapeptide of formula Ia is prepared by a process comprising the step of condensing an activated compound of formula IIa with a tripeptide of formula IIIa

16. The process according to any one of the preceding claims, wherein the tetrapeptide of formula I contains less than 0.5% of histidine D-isomer impurity as determined by HPLC, preferably less than 0.4% of histidine D-isomer impurity as determined by HPLC.

17. The method according to any one of the preceding claims, wherein the tetrapeptide of formula I has a purity of 98% or more as determined by HPLC, preferably a purity of 99% or more as determined by HPLC.

18. A method for preparing liraglutide or a pharmaceutically acceptable salt thereof, comprising converting the tetrapeptide of formula I obtained by the method according to any one of the preceding claims into liraglutide or a pharmaceutically acceptable salt thereof.

19. Liraglutide or a pharmaceutically acceptable salt thereof obtained by the method according to claim 18.

20. Liraglutide or a pharmaceutically acceptable salt thereof according to claim 19, which contains less than 0.5% of histidine D-isomer impurity as determined by HPLC, preferably less than 0.4% of histidine D-isomer impurity as determined by HPLC.