Method for preparing tetrapeptide fragment of liraglutide

Abstract

The present invention relates to an improved process for the preparation of a tetrapeptide of formula (I) wherein R1 and R2 are independently selected from amine protecting groups and R3 is t-Bu or Bn. The invention further relates to a process for preparing liraglutide or a pharmaceutically acceptable salt thereof using the tetrapeptide of formula (I). # imgabs0 #

Description

[0001] Cross-references to Related Patent Applications

[0002] This patent application claims priority to Indian Patent Application No. 202311008660 filed on February 10, 2023, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention relates to an improved process for preparing a tetrapeptide of formula I, which is used to synthesize liraglutide.

[0004]

[0005] wherein R1 and R2 are independently selected from amine protecting groups, and R3 is t-Bu or Bn.

[0006] The present invention further relates to a method for preparing liraglutide or a pharmaceutically acceptable salt thereof using the tetrapeptide of formula I prepared by the method of the present invention. Background Art

[0007] Liraglutide is a human GLP-1 receptor agonist (or GLP-1 analog) represented by the following formula:

[0008]

[0009] Liraglutide (brand name Liraglutide (marketed by Novo Nordisk) is an antidiabetic medication used to treat type 2 diabetes, obesity, and long-term weight management. Liraglutide was approved for medical use in the European Union in 2009 and in the United States in 2010.

[0010] Liraglutide and its preparation have been disclosed in US6268343.

[0011] Typically, liraglutide is prepared by chemical synthesis, either by solid phase synthesis or by sequential coupling of amino acids in solution phase, or by convergent synthesis involving the coupling of separately synthesized fragments.

[0012] Most methods known in the art for synthesizing liraglutide involve synthesizing a variety of short-chain peptide fragments in solution phase or solid phase, such as dipeptides, tripeptides, tetrapeptides, pentapeptides, etc. These fragments are then coupled in solid phase or solution phase or by solid-liquid mixing methods to provide liraglutide.

[0013] The tetrapeptide fragment of formula I is one such fragment used in the synthesis of liraglutide.

[0014] Various methods are known in the art for the synthesis of tetrapeptide fragments of formula I with suitable protecting groups in both solid and liquid phases.

[0015] WO2016046753A1 discloses a method for synthesizing a tetrapeptide fragment, wherein the tetrapeptide fragment is prepared by solid phase synthesis, and the purity of the tetrapeptide fragment is 94% as determined by HPLC.

[0016] CN102875665B and CN106478805B also disclose the solid phase synthesis of the tetrapeptide of formula I, wherein the purity of the tetrapeptide is about 94% and 96.6%, respectively, as determined by HPLC.

[0017] However, solid phase synthesis of short-chain peptides, such as the tetrapeptide of Formula I, is not economically and commercially feasible due to the large amounts of solvent used in the process. Recovery of the final product is also cumbersome due to the multiple resin washes, elutions, and the use of large amounts of solvent to purify the product. Scale-up of the batch is also limited. Consequently, solid phase synthesis of short-chain peptide fragments is generally not favored because the process is not worth the effort involved in the synthesis.

[0018] CN105732798B discloses another method for synthesizing a tetrapeptide fragment of formula I. The disclosed method involves linear coupling of histidine (His) and alanine (Ala), glutamic acid (Glu) and glycine (Gly) in the presence of a solvent and a base.

[0019] The tetrapeptide of formula I contains histidine at the amino terminus. Histidine is an amino acid that is highly susceptible to racemization. When exposed to alkali, the tendency of histidine to racemize increases.

[0020] The method disclosed in CN105732798B results in the histidine amino acid being exposed to alkali during two stages of the synthesis, which leads to racemization of the histidine and the formation of a D-isomer histidine impurity in the tetrapeptide. When the tetrapeptide with a high D-isomer histidine impurity is converted into liraglutide, the impurity is further carried over into the final API stage. This impurity is extremely difficult to separate from the final peptide, and the final peptide can contain varying amounts of D-His impurity. The resulting liraglutide product has a high level of D-isomer impurity, making it unsuitable for pharmaceutical formulations.

[0021] As is apparent from the above, the reported methods for preparing liraglutide tetrapeptide fragments require complicated operating conditions and fail to provide products of high purity, especially with respect to the D-isomer histidine impurity.

[0022] Various methods disclosed in the prior art also involve cumbersome work procedures and multiple purification steps, and are not cost-effective.

[0023] Therefore, there is still a need to develop an efficient, simple and industrially feasible synthetic method that can overcome the shortcomings of the prior art and provide highly pure tetrapeptide fragments of Formula I and liraglutide in a cost-effective manner.

[0024] Purpose of the Invention

[0025] An object of the present invention is to overcome the above-mentioned disadvantages of the prior art.

[0026] Another object of the present invention is to provide an improved, commercially viable method for synthesizing the tetrapeptide fragment of formula I, and further a method for synthesizing liraglutide using the tetrapeptide fragment of formula I prepared by the method described in the present invention.

[0027] A further object of the present invention is to obtain highly pure tetrapeptide fragments of formula I and liraglutide or pharmaceutically acceptable salts thereof. Summary of the Invention

[0028] The present invention relates to an improved process for preparing a tetrapeptide of formula I, which is used in the synthesis of liraglutide.

[0029] In one aspect, the present invention relates to an improved process 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,

[0030]

[0031] The method comprises the steps of condensing an activated compound of formula II with a tripeptide of formula III,

[0032]

[0033] wherein R1 and R2 are as defined above, and A is an acid-activating group,

[0034]

[0035] wherein R3 is as defined above.

[0036] In a further aspect, the present invention relates to a method for preparing liraglutide or a pharmaceutically acceptable salt thereof, comprising converting the tetrapeptide of formula I obtained by the method of the present invention into liraglutide or a pharmaceutically acceptable salt thereof.

[0037] definition

[0038] Unless the context indicates otherwise, the following definitions apply in connection with this application.

[0039] "Peptide" refers to a short chain of amino acids in which two or more amino acids are chemically linked by amide bonds.

[0040] "Dipeptide" refers to a peptide having a chain of two amino acids.

[0041] "Tripeptide" refers to a peptide having a chain of three amino acids.

[0042] "Tetrapeptide" refers to a peptide having a chain of four amino acids.

[0043] The term "fragment" refers to a sequence of two or more amino acids. The amino acids in the fragment may be protected or unprotected.

[0044] The term "condensation" refers to a condensation reaction by the reaction of a carboxyl group with an amine group to form an amide bond.

[0045] The term "carboxylic acid" refers to an organic compound containing a -COOH group.

[0046] The term "amine" refers to an organic compound containing an -NH2 group.

[0047] The term "activating compound" refers to a compound having an activated ester group.

[0048] The term "acid activating group" refers to a group that increases the reactivity of a carboxyl group. Activated carboxylic acids undergo the same reactions as their unactivated analogs, but react faster.

[0049] The term "activated ester" refers to an ester functional group that is highly susceptible to nucleophilic attack. Activated esters undergo the same reactions as their unactivated analogs, but react more rapidly. Activated esters can be prepared by reacting the hydroxyl (–OH) group of a carboxylic acid with an activating agent to convert it into a favorable leaving group.

[0050] The term "leaving group" refers to an atom, group of atoms, or fragment that detaches from the main or remaining portion of a substrate during a reaction or an essential step in a reaction.

[0051] The term "activator" refers to a compound that activates a molecule or group by introducing an activating group into the molecule, thereby activating the molecule or group to react with another group or molecule.

[0052] The term "coupling agent" refers to a reagent that promotes the formation of a bond between two adjacent groups.

[0053] The term "protecting group" refers to a group 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 synthetic conditions of one or more subsequent steps to which the molecule is subjected, while allowing removal of the protecting group under conditions that do not harm the remaining molecule.

[0054] The term "amine protecting group" refers specifically to a protecting group attached to an amine functional group in a molecule.

[0055] For the purposes of this invention, in a molecule having two or more amine protecting groups, the "amine protecting groups" can be the same or different from each other.

[0056] The protecting group can be cleaved from the molecule after the desired compound is obtained.The term "deprotection" refers to the cleavage or removal of a protecting group.

[0057] Protecting groups can be deprotected under acidic, basic and / or neutral conditions. Protection and deprotection methods are known in the art (see, in particular, "Protective groups in organic synthesis", Greene TW and Wuts P. GM, Wiley-Interscience, 1999).

[0058] The term "ambient temperature" refers to a temperature in the range of about 15°C to 35°C. Detailed Description of the Invention

[0060] The present invention relates to a process for preparing a tetrapeptide of formula I,

[0061]

[0062] wherein R1 and R2 are independently selected from amine protecting groups, and R3 is t-Bu or Bn.

[0063] In some embodiments, the amine protecting group is selected from the group consisting of tert-butyloxycarbonyl, trityl, 4-methyltrityl, monomethoxytrityl, fluorenylmethoxycarbonyl, carboxybenzyl, N-benzyloxymethyl, and tosyl.

[0064] In one embodiment, R1 and R2 are the same amine protecting group.

[0065] In another embodiment, R1 and R2 are different amine protecting groups.

[0066] In a preferred embodiment, R1 is selected from tert-butyloxycarbonyl, trityl, 4-methyltrityl, monomethoxytrityl, carboxybenzyl, fluorenylmethoxycarbonyl.

[0067] In another preferred embodiment, R2 is selected from the group consisting of trityl, 4-methyltrityl, monomethoxytrityl, N-benzyloxymethyl, fluorenylmethyloxycarbonyl, tosyl and tert-butyloxycarbonyl.

[0068] In a more preferred embodiment, R1 is tert-butyloxycarbonyl and R2 is selected from trityl and 4-methyltrityl.

[0069] In an even more preferred embodiment, R1 is tert-butyloxycarbonyl and R2 is trityl.

[0070] In one aspect, the method comprises the step of condensing an activated compound of formula II with a tripeptide of formula III,

[0071]

[0072] wherein R1 and R2 are as defined above; A is an acid-activated group,

[0073]

[0074] wherein R3 is as defined above.

[0075] In one embodiment, the acid-activated 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.

[0076] 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.

[0077] The base may be selected from the group consisting of N,N-diisopropylethylamine, triethylamine, methylmorpholine, sodium bicarbonate, sodium carbonate and potassium carbonate, preferably triethylamine.

[0078] The solvent may be 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. Preferably, the solvent is dichloromethane.

[0079] In yet another embodiment, the condensation reaction is carried out at ambient temperature, preferably at a temperature of 20°C to 35°C, more preferably at a temperature of 20°C to 30°C.

[0080] In a preferred embodiment, the tetrapeptide of Formula I is a compound of Formula Ia [Formula I, wherein R1 is Boc, R2 is Trt, and R3 is t-Bu].

[0081]

[0082] In a preferred embodiment, the present invention relates to a process for preparing a compound of formula Ia, which comprises condensing an activated compound of formula IIa [Formula II, wherein R1 is Boc, R2 is Trt and A is ONB] with a tripeptide of formula IIIa [Formula III, wherein R3 is t-Bu].

[0083]

[0084] In another preferred embodiment, the condensation is carried out in the 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 Ia is isolated from the reaction mixture after extraction with dichloromethane and precipitation with ethyl acetate.

[0085] In another embodiment, the activated compound of formula II is prepared by activating the carboxyl group of the compound of formula IV using an activating agent,

[0086]

[0087] wherein R1 and R2 are as defined above.

[0088] 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.

[0089] In one embodiment, activation of the carboxyl group is carried out in the presence of a coupling agent in a solvent.

[0090] The coupling agent can be selected from the group consisting of: N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, N,N-dicyclohexylcarbodiimide, Oxyma B / diisopropylcarbodiimide, benzotriazole-1-yl-oxy-tris-pyrrolidinyl-phosphonium hexafluorophosphate, azabenzotriazole tetramethyl uronium hexafluorophosphate, O-(1H-benzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate and propyl phosphoric anhydride. Preferably, the coupling agent is selected from the group consisting of: N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, N,N-dicyclohexylcarbodiimide and Oxyma B / diisopropylcarbodiimide. More preferably, the coupling agent is N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride.

[0091] The solvent may be selected from the group consisting of dichloromethane, 1-methyl-pyrrolidone-2-one, acetonitrile, tetrahydrofuran, dimethyl sulfoxide, N,N-dimethylformamide, N-methylformamide, dimethyl carbonate, diethyl carbonate and mixtures thereof. Preferably, the activation of the carboxyl group using an activating agent is carried out in tetrahydrofuran in the presence of a coupling agent.

[0092] In a preferred embodiment, the compound of Formula IV (wherein R1 is Boc and R2 is Trt) is activated using N-hydroxy-5-norbornene-2,3-dicarboximide (HONB) to form the activated compound of Formula IIa [Formula II, where R1 is Boc, R2 is Trt and A is ONB].

[0093] In another preferred embodiment, the compound of formula IV is treated with N-hydroxy-5-norbornene-2,3-dicarboximide (HONB) in tetrahydrofuran at a temperature of 20-30° C. for 10 to 12 hours in the presence of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride. After the reaction is complete, the activated compound of formula II is isolated by adding water and dichloromethane.

[0094] In one embodiment, the present invention relates to a process for preparing a tripeptide of formula III, comprising the step of activating the carboxyl group of a compound of formula V using an activating agent in the presence of a coupling agent in a solvent to obtain an activated compound of formula VI,

[0095]

[0096] Wherein R4 is an amine protecting group,

[0097]

[0098] wherein R4 and A are as defined above.

[0099] The amine protecting group (R4) may be selected from fluorenylmethyloxycarbonyl, tert-butyloxycarbonyl, carboxybenzyl and toluenesulfonyl, and preferably it is fluorenylmethyloxycarbonyl.

[0100] 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.

[0101] In one embodiment, activation of the carboxyl group is performed in the presence of a coupling agent in a solvent.

[0102] The coupling agent can be selected from the group consisting of: N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, N,N-dicyclohexylcarbodiimide, Oxyma B / diisopropylcarbodiimide, benzotriazole-1-yl-oxy-tris-pyrrolidinyl-phosphonium hexafluorophosphate, azabenzotriazole tetramethyl uronium hexafluorophosphate, O-(1H-benzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate and propyl phosphoric anhydride. Preferably, the coupling agent is selected from the group consisting of: N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, N,N-dicyclohexylcarbodiimide and Oxyma B / diisopropylcarbodiimide. More preferably, the coupling agent is N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride.

[0103] The solvent may be selected from the group consisting of dichloromethane, 1-methyl-pyrrolidone-2-one, acetonitrile, tetrahydrofuran, dimethyl sulfoxide, N,N-dimethylformamide, N-methylformamide, dimethyl carbonate, diethyl carbonate and mixtures thereof. Preferably, the activation of the carboxyl group using an activating agent is carried out in tetrahydrofuran in the presence of a coupling agent.

[0104] In a preferred embodiment, the compound of formula V (wherein R4 is Fmoc) is activated using N-hydroxy-5-norbornene-2,3-dicarboximide (HONB) to generate the activated compound of formula VI (wherein R4 is Fmoc; A is ONB).

[0105] In another preferred embodiment, the compound of formula V is treated with N-hydroxy-5-norbornene-2,3-dicarboximide (HONB) in the presence of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride in tetrahydrofuran at 25°C to 30°C for 10 to 12 hours to produce the activated compound of formula VI. After the reaction is complete, the activated compound of formula VI is isolated in dichloromethane by adding water and dichloromethane.

[0106] In one embodiment, the activated compound of formula VI is further condensed with a dipeptide of formula VII in the presence of a base in a solvent to obtain a protected tripeptide of formula VIII,

[0107]

[0108] Wherein R3 is t-Bu or Bn,

[0109]

[0110] wherein R3 is t-Bu or Bn and R4 is an amine protecting group.

[0111] The amine protecting group (R4) may be selected from fluorenylmethyloxycarbonyl, tert-butyloxycarbonyl, carboxybenzyl and toluenesulfonyl, and preferably it is fluorenylmethyloxycarbonyl.

[0112] 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.

[0113] The solvent may be 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. Preferably, the solvent is dichloromethane.

[0114] In a preferred embodiment, an activated compound of formula VI (wherein R4 is Fmoc; A is ONB) is condensed with a dipeptide of formula VII (wherein R3 is t-Bu) to provide a protected tripeptide of formula VIII (wherein R3 is t-Bu and R4 is Fmoc).

[0115] In another preferred embodiment, the condensation is carried out in the presence of triethylamine and using dichloromethane as solvent.

[0116] The protected tripeptide of formula VIII is deprotected by any method known in the art to obtain the tripeptide of formula III. The deprotection is carried out in acid or base, depending on the protecting group to be removed.

[0117] Preferably, the protected tripeptide of formula VIII is deprotected in the presence of an organic base in a solvent.

[0118] 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.

[0119] 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-dimethylformamide, N-methylformamide, dimethyl carbonate, diethyl carbonate and mixtures thereof. Preferably, the solvent is dichloromethane.

[0120] More preferably, the deprotection of the protected tripeptide of formula VIII can be carried out in the presence of diethylamine in dichloromethane solvent.

[0121] Preferably, 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 certain embodiments, the solid is isolated by adding diisopropyl ether, filtering and drying.

[0122] The dipeptide of formula VII is obtained by a process comprising the steps of activating the carboxyl group of a compound of formula IX using an activating agent to provide a compound of formula X,

[0123]

[0124] Wherein R5 is an amine protecting group,

[0125]

[0126] wherein R5 and A are as defined above.

[0127] The amine protecting group R5 may be selected from the group consisting of fluorenylmethyloxycarbonyl, tert-butyloxycarbonyl, carboxybenzyl and toluenesulfonyl; preferably it is fluorenylmethyloxycarbonyl.

[0128] The activation of formula IX is carried out by an activating agent in the presence of a coupling agent and a solvent.

[0129] 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.

[0130] In one embodiment, activation of the carboxyl group is performed in the presence of a coupling agent in a solvent.

[0131] The coupling agent can be selected from the group consisting of: N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, N,N-dicyclohexylcarbodiimide, Oxyma B / diisopropylcarbodiimide, benzotriazole-1-yl-oxy-tris-pyrrolidinyl-phosphonium hexafluorophosphate, azabenzotriazole tetramethyl uronium hexafluorophosphate, O-(1H-benzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate and propyl phosphoric anhydride. Preferably, the coupling agent is selected from the group consisting of: N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, N,N-dicyclohexylcarbodiimide and Oxyma B / diisopropylcarbodiimide. More preferably, the coupling agent is N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride.

[0132] The solvent may be selected from the group consisting of dichloromethane, 1-methyl-pyrrolidone-2-one, acetonitrile, tetrahydrofuran, dimethyl sulfoxide, N,N-dimethylformamide, N-methylformamide, dimethyl carbonate, diethyl carbonate and mixtures thereof. Preferably, the activation of the carboxyl group using an activating agent is carried out in tetrahydrofuran in the presence of a coupling agent.

[0133] In a preferred embodiment, the compound of formula IX (wherein R5 is Fmoc) is activated using N-hydroxy-5-norbornene-2,3-dicarboximide (HONB) to generate the activated compound of formula X (wherein R5 is Fmoc; A is ONB).

[0134] In a preferred embodiment, the compound of formula IX is treated with N-hydroxy-5-norbornene-2,3-dicarboximide (HONB) in the presence of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride in tetrahydrofuran at 20-30° C. for 6 to 8 hours, resulting in activation of the compound of formula IX. After the reaction is complete, the obtained compound of formula X is extracted into dichloromethane by adding water and dichloromethane.

[0135] The starting compound of formula IX (wherein R5 is Fmoc) can be obtained from commercial sources.

[0136] 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.

[0137] 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).

[0138] The protected dipeptide obtained above is deprotected by any method known in the art to obtain the dipeptide of formula VII. The deprotection is carried out in acid or base, depending on the protecting group to be removed.

[0139] Preferably, the protected dipeptide is reacted with an organic base in a solvent.

[0140] 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.

[0141] In the presence of an organic base, the solvent used for the deprotection reaction can be 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. Preferably, the solvent is dichloromethane.

[0142] 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°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 adding water and dichloromethane, then filtering and drying to produce the dipeptide of Formula VII.

[0143] In some embodiments, the activated ester compounds of Formula II, VI, and X obtained in the methods of the present invention are prepared in situ and are not isolated during the process.

[0144] In the method of the present invention, the tetrapeptide of Formula I is prepared by using histidine in the final stage of the reaction sequence. This limits the exposure of the histidine to base during the process. Using the method described herein, exposure of the histidine residue / histidine-containing peptide to base occurs only once during the synthesis, so racemization of the histidine to the D-isomer is minimal.

[0145] Surprisingly, the inventors found that following the order of reaction steps described in the method of the present application and sequentially condensing amino acids and fragments, such as introducing histidine in the final stage, greatly reduced the histidine D-isomer impurity in the tetrapeptide fragment.

[0146] In contrast, the prior art method discloses the introduction of histidine in the first stage, whereby the histidine residue is exposed to base at each stage in the subsequent reaction steps until the tetrapeptide formation is complete. This results in the racemization of histidine during the process, resulting in a product with a high content of impurities, particularly a high content of D-histidine impurity.

[0147] The inventors of the present invention also attempted to synthesize the tetrapeptide of Formula I by introducing histidine in the second stage rather than the first stage to reduce the exposure of histidine to base during the process. This also resulted in increased racemization of histidine, resulting in a D-His impurity of approximately 3%, while the process of the present invention produces a tetrapeptide fragment with a negligible content of histidine D-isomer, without any further purification. The tetrapeptide of Formula I obtained by the process of the present invention 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, more preferably less than 0.3% of histidine D-isomer impurity as determined by HPLC, and even more preferably less than 0.2% of histidine D-isomer impurity as determined by HPLC.

[0148] The method according to the present invention results in a tetrapeptide of Formula I having an extremely low content of the histidine D-isomer impurity (e.g., less than 0.5% as determined by HPLC) even in the crude tetrapeptide. Furthermore, the crude product can 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 does not need to be used to remove D-His impurities, which are already controlled in the crude product obtained by using the method according to the present invention.

[0149] In some embodiments, the tetrapeptide of Formula I is purified using a solvent selected from the group consisting of ethyl acetate, acetone, isopropanol, tetrahydrofuran, acetonitrile, and mixtures thereof.

[0150] The purity of the tetrapeptide of formula I obtained by the process of the present invention is 98% or higher as determined by HPLC, preferably 99% or higher as determined by HPLC, most preferably 99.4% as determined by HPLC.

[0151] Preferably, the method of the present invention is carried out in solution phase. This also eliminates the disadvantages of solid phase synthesis, such as the use of expensive resins, multiple washes with solvents, and problems with batch scale-up.

[0152] In a further embodiment, the tetrapeptide of formula I obtained by the method of the present invention can be converted into liraglutide or a pharmaceutically acceptable salt thereof by methods known in the art, for example, using the method reported in WO2016046753A1.

[0153] The use of the highly pure tetrapeptide fragment of formula I obtained by the process of the present invention positively influences the yield and purity of liraglutide or a salt thereof.

[0154] Therefore, the present invention provides liraglutide or a pharmaceutically acceptable salt thereof, 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, more preferably less than 0.3% of histidine D-isomer impurity as determined by HPLC, even more preferably less than 0.2% of histidine D-isomer impurity as determined by HPLC.

[0155] Therefore, the inventors of the present invention have developed an improved method for synthesizing not only tetrapeptide fragments but also liraglutide or a pharmaceutically acceptable salt thereof, which method is both cost-effective and commercially viable.

[0156] The method can be efficiently scaled up and the products or their intermediates at various stages of the synthesis can be separated using separation techniques such as solvent extraction and solvent recovery, precipitation, distillation, filtration, and product drying.

[0157] Therefore, the synthesis method of the present invention improves the purity of the peptide, reduces the cost of raw materials and purification, and is conducive to industrial production.

[0158] Abbreviations / Acronyms:

[0159] HONB: N-hydroxy-5-norbornene-2,3-dicarboximide, respectively ONB refers to the same molecule when it is linked to a second molecule via a hydroxyl group (so that there is no H on the hydroxyl group).

[0160] EDC.HCl: N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride

[0161] DCC: dicyclohexylcarbodiimide

[0162] Boc: tert-butyloxycarbonyl

[0163] Fmoc: fluorenylmethoxycarbonyl

[0164] Trt: trityl

[0165] Bn-: benzyl-

[0166] His:histidine

[0167] Ala: alanine

[0168] Glu: glutamate

[0169] Gly: glycine

[0170] t-Bu: tert-butyl

[0171] OH: hydroxyl

[0172] D-isomer: right-handed isomer

[0173] HPLC: High Performance Liquid Chromatography DETAILED DESCRIPTION

[0174] experiment

[0175] Detailed experimental parameters for the present invention are provided by the following examples, which are intended to illustrate but not to limit all possible embodiments of the present invention.

[0176] Example:

[0177] Comparative Example

[0178] Preparation of tetrapeptide: (Boc)-His(Trt)-Ala-Glu(OtBu)-Gly-OH)

[0179] Stage-1: Preparation of dipeptide: Glu(OtBu)-Gly-OH

[0180]

[0181] a) EDC.HCl (101.4 g) is added to a mixture of HONB (94.78 g) and Fmoc-Glu(OtBu)-OH (150 g) in tetrahydrofuran (1400 ml). The mixture is stirred at 20-30° C. for 15-16 hours. Distilled water (750 ml) and brine solution (150 ml) are added to the reaction mixture and stirred for 190 minutes, after which it is allowed to stand. The aqueous layer is separated and discarded. Water (750 ml) and brine solution (150 ml) are added to the organic layer. The mixture is stirred. The layers are separated and the aqueous layer is discarded.

[0182] b) Triethylamine solution (35.7 g dissolved in 150 ml of tetrahydrofuran) and glycine solution (26.5 g dissolved in 300 ml of distilled water) are slowly added to the organic layer at 20-30°C and the reaction mixture is stirred for 3 to 5 hours. Distilled water (600 ml) and ethyl acetate (600 ml) are added to the reaction mixture and the pH is adjusted to 3 to 4. The reactants are stirred at 20-30°C for 10 minutes. The mixture is allowed to stand and the layers are separated. Sodium bicarbonate solution (52.5 g dissolved in 750 ml of distilled water) and brine solution (150 ml) are added to the organic layer and the mixture is stirred at 20-30°C for 15 minutes, then allowed to stand and the layers are separated. Distilled water is added to the organic layer and the pH of the reactants is adjusted to 3 to 4. The reactants are stirred for 10 minutes, then allowed to stand and the layers are separated. The aqueous layer is discarded and the organic layer is recovered under vacuum at a temperature below 40°C. The residue was degassed under vacuum for 120 minutes and dichloromethane (300 ml) was added to the residue. The mixture was stirred to obtain a clear solution and heated to 35 to 40° C. The solid was isolated from the reaction mixture by adding diisopropyl ether, cooling, filtering and drying under vacuum.

[0183] c) Dichloromethane (450 ml) and Fmoc-Glu(OtBu)-Gly (90 gm) were stirred at 20-30°C for 5-10 minutes. Diethylamine (90 ml) was added, and the resulting mixture was stirred at 20-30°C 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 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°C. Dichloromethane (300 ml) was added to the residue, and the residue was recovered under vacuum at a temperature below 45°C. The residue was degassed under vacuum at 35-45°C for 1-2 hours. A solid (44.5 g) was isolated by adding dichloromethane, followed by filtration, washing with dichloromethane, and drying under vacuum at a temperature of 40-50°C for 14-16 hours.

[0184] Yield: 48.5% (purity: 98.2%).

[0185] Stage-2: Preparation of dipeptide: Boc-His(Trt)-Ala-OH

[0186]

[0187] 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 at 20-30°C overnight until the reaction was complete. The reaction was cooled to 0-10°C and L-alanine solution (9 g dissolved in 125 ml of deionized water) and triethylamine solution (20.4 gm dissolved in 50 ml of 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 dissolved in 1000 ml of 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 is washed with distilled water, distilled at 40-50 ℃, and degassed. Acetonitrile (500ml) is added to the resulting material and heated to 40-50 ℃ for 30 minutes, then cooled to 35-40 ℃. Dichloromethane (50ml) is added to the reaction mixture and stirred for 1 hour, then cooled to 20-25 ℃. The reaction mixture is stirred at 20-30 ℃ for 4 hours, then filtered solid and washed with acetonitrile (100ml) and n-hexane (100ml). The reaction mixture is filtered and dried in a vacuum to obtain solid (42g).

[0188] Yield: 73.4%.

[0189] Stage-3: Preparation of tetrapeptide: (Boc)-His(Trt)-Ala-Glu(OtBu)-Gly-OH)

[0190]

[0191] 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 at 20-30° C. overnight until the reaction was complete. The reaction was cooled to 0-5° C., 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° C. Triethylamine (5 ml) was added and the reaction was stirred at 20-30° C. for 2 hours until the reaction was complete. The reaction was evaporated at 35-40° C. under vacuum and the obtained residue 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 is added to the organic layer, and the pH is adjusted to 4.0-4.1 with hydrochloric acid solution. The organic layer is separated and evaporated at 35-40 ℃ under vacuum. Water and dichloromethane (50ml) are added to the reactant and stirred. Isolate the dichloromethane layer and slowly add it to diisopropyl ether (150ml). Filter the precipitate, wash with diisopropyl ether, and dry under vacuum to obtain a solid (6g).

[0192] Yield: 42.07% (purity: 85.1%); D-isomer: 2.99%.

[0193] Example-1

[0194] Preparation of tetrapeptide: (Boc)-His(Trt)-Ala-Glu(OtBu)-Gly-OH)

[0195] Stage-1: Preparation of dipeptide: Glu(OtBu)-Gly-OH

[0196]

[0197] a) EDC-HCl (101.4 g) is added to a mixture of HONB (94.78 g) and Fmoc-Glu(OtBu)-OH (150 g) in tetrahydrofuran (1400 ml). The mixture is stirred at 20-30° C. for 15-16 hours. Distilled water (750 ml) and brine solution (150 ml) are added to the reaction mixture and stirred for 190 minutes, after which it is allowed to stand. The aqueous layer is separated and discarded. Water (750 ml) and brine solution (150 ml) are added to the organic layer. The mixture is stirred. The layers are separated and the aqueous layer is discarded.

[0198] b) Triethylamine solution (35.7 g dissolved in 150 ml of tetrahydrofuran) and glycine solution (26.5 g dissolved in 300 ml of distilled water) are slowly added to the organic layer at 20-30°C and the reaction mixture is stirred for 3 to 5 hours. Distilled water (600 ml) and ethyl acetate (600 ml) are added to the reaction mixture and the pH is adjusted to 3 to 4. The reactants are stirred at 20-30°C for 10 minutes. The mixture is allowed to stand and the layers are separated. Sodium bicarbonate solution (52.5 g dissolved in 750 ml of distilled water) and brine solution (150 ml) are added to the organic layer and the mixture is stirred at 20-30°C for 15 minutes, then allowed to stand and the layers are separated. Distilled water is added to the organic layer and the pH of the reactants is adjusted to 3 to 4. The reactants are stirred for 10 minutes, then allowed to stand and the layers are separated. The aqueous layer is discarded and the organic layer is recovered under vacuum at a temperature below 40°C. The residue was degassed under vacuum for 120 minutes and dichloromethane (300 ml) was added to the residue. The mixture was stirred to obtain a clear solution and heated to 35 to 40° C. The solid was isolated from the reaction mixture by adding diisopropyl ether, cooling, filtering and drying under vacuum.

[0199] c) Dichloromethane (450 ml) and Fmoc-Glu(OtBu)-Gly (90 gm) were stirred at 20-30°C for 5-10 minutes. Diethylamine (90 ml) was added and the resulting mixture was stirred at 20-30°C 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°C. Dichloromethane (300 ml) was added to the residue and recovered under vacuum at a temperature below 45°C. The residue was degassed under vacuum at 35-45°C for 1 to 2 hours. A solid (44.5 g) was isolated by adding dichloromethane, followed by filtration, washing with dichloromethane, and drying under vacuum at a temperature of 40-50°C for 14-16 hours.

[0200] Yield: 48.5% (purity: 98.2%).

[0201] Stage-2: Preparation of dipeptide: Ala-Glu(OtBu)-Gly-OH

[0202]

[0203] 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° C. for 10 to 12 hours. After the reaction was complete, distilled water and dichloromethane (500 ml) were added to the reaction mixture and stirred for 10-15 minutes, then allowed to stand. The aqueous layer was separated and the product was extracted with dichloromethane. The dichloromethane layers were combined and water was added. The layers were separated and the aqueous layer was discarded. The organic layer was retained.

[0204] b) Glu(OtBu)-Gly-OH (83.59 g) was added to the dichloromethane layer at 20-30° C. 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° C. Methanol and distilled water were added to the residue and the reaction mixture was cooled to 20-30° C. 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 extracting with methanol at a temperature of 35 to 45° C. and cooling to 20-30° C.

[0205] c) Dichloromethane (500 ml) and diethylamine (100 ml) are added to the solid (Fmoc-Ala-Glu(OtBu)-Gly-OH) obtained in step b) at 20-30° C. The reaction is stirred for 5-8 hours and distilled water is added. The layers are separated and the aqueous layer is recovered under vacuum at below 50° C. Dichloromethane (500 ml) is added to the residue and the mixture is heated to 35-45° C. A solid (71 g) is isolated from the reaction mixture by adding diisopropyl ether, filtering and drying under vacuum at 40-50° C. for 15-18 hours.

[0206] Yield: 66.7% (purity: 94.8%).

[0207] Stage-3: Preparation of tetrapeptide: (Boc)-His(Trt)-Ala-Glu(OtBu)-Gly-OH)

[0208]

[0209] 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° C. for 10 to 12 hours. After the reaction was complete, distilled water and dichloromethane (500 ml) were added to the reaction mixture, stirred for 10-15 minutes, and then allowed to stand.

[0210] b) Ala-Glu(OtBu)-Gly-OH (66.57 g) is added to the dichloromethane layer from the above step, and triethylamine (30.5 g) and distilled water (50 ml) are added thereto. The reaction is stirred at 20-30° C. for 2 to 4 hours, and the pH of the reaction is adjusted to 3.0 to 4.0. The layers are separated. The aqueous layer is further washed with dichloromethane. The dichloromethane layers are combined, and saturated sodium bicarbonate solution (500 ml) is added thereto and stirred for 10-15 minutes. The reaction mixture is allowed to stand and the layers are separated. The organic layer is recovered under vacuum at a temperature below 45° C. to obtain a residue (crude product; D-isomer: 0.34%).

[0211] Ethyl acetate (500 ml) was added to the residue and the temperature was raised to 35-45° C. The reaction was stirred at 35-45° C. for 2-3 hours and cooled to a temperature of 20-30° C. The solid (118 g) was isolated by filtration and washed with ethyl acetate and dried under vacuum at 40-45° C. for 15-18 hours.

[0212] Yield: 72.4% (purity 99.4%); D-isomer: 0.22%.

[0213] Example-2

[0214] Preparation of tetrapeptide: (Boc)-His(Trt)-Ala-Glu(OtBu)-Gly-OH)

[0215] Stage-1: Preparation of dipeptide: Glu(OtBu)-Gly-OH

[0216]

[0217] a) EDC. Stir the mixture at 20-30°C for 12-15 hours. Cool the reaction mixture to 10-15°C. Add pre-cooled distilled water (1000 ml) and cold saline solution to the reaction mixture and stir. Allow to stand, separate the layers, and discard the aqueous layer. Wash the organic layer with pre-cooled distilled water (1000 ml) and cold saline solution. Allow the mixture to stand again, and discard the aqueous layer.

[0218] b) Add the glycine solution and triethylamine solution to the organic layer at 10-20°C. Stir the reaction mixture for 4-6 hours, then cool the reaction mixture to 10-15°C. Add pre-cooled distilled water (800 ml) and pre-cooled ethyl acetate (2000 ml) at 10-15°C. Adjust the pH to 3 to 3.5. Stir the reaction mixture at 10-15°C for 10-15 minutes, then let the layers stand and discard the aqueous layer. Add pre-cooled sodium bicarbonate solution and cold saline solution to the reaction mixture (organic layer) at 10-20°C. Stir the reaction mixture and separate the layers. Add pre-cooled distilled water (1000 ml) to the organic layer at 10-15°C. Adjust the pH to 3 to 3.5 and stir the reaction mixture for 10-15 minutes. Let stand and separate the layers, discarding the aqueous layer. Recover the organic layer under vacuum at a temperature below 50°C. Add dichloromethane (400ml) and then recover under vacuum at a temperature below 50°C. The residue is degassed under vacuum and dichloromethane (400ml) is added thereto. Stir to obtain a clear solution. The reactant is heated to 35-40°C and diisopropyl ether (2000ml) is added, and the reaction mixture is stirred at 35-45°C for 30-60 minutes. The reaction mixture is cooled to 20-30°C and stirred for 2-3 hours, and the obtained solid is filtered. The solid is washed with diisopropyl ether (200ml) and dried under vacuum for 100-120 minutes and used as is in the next step.

[0219] Dichloromethane (400 ml) was added to the residue and stirred to obtain a clear solution. The reaction mixture was heated to 35-40° C. and diisopropyl ether (200 ml) was added thereto. The mixture was stirred at 35-45° C. for 30-60 minutes and cooled to 20-30° C. The mixture was stirred for 2-3 hours and the obtained solid was filtered, washed with diisopropyl ether and dried under vacuum.

[0220] c) Dichloromethane (1000 ml) is added to the residual solid and the reaction mixture is stirred at 20-30°C for 5-10 minutes. Diethylamine (200 ml) is added thereto and the mixture is stirred for 8-10 hours. Distilled water (1000 ml) is added to the reaction mixture and stirred for 20-25 minutes. The layers are separated. The aqueous layer is washed with dichloromethane and distilled under vacuum at a temperature below 50°C. Dichloromethane (400 ml) is added thereto and then recovered under vacuum at a temperature below 40°C. Dichloromethane (1000 ml) is added to the residue and heated to 30-40°C. Then diisopropyl ether (1000 ml) is added to the reactant at 30-40°C and stirred for 60 minutes. The reaction mixture is cooled to 20 to 30°C and stirred for 2-3 hours. The solid (74 g) is isolated by filtration, washing with dichloromethane and drying under vacuum.

[0221] Yield: 60.6% (purity 99.87%)

[0222] Stage-2: Preparation of tripeptide: Ala-Glu(OtBu)-Gly-OH

[0223]

[0224] 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° C. for 12-15 hours. The reaction mixture was cooled to 10-15° C. Precooled distilled water (400 ml) and cold brine solution were added to the reaction mixture and stirred.

[0225] The layers were allowed to settle and the aqueous layer was discarded. The organic layer was washed with pre-chilled distilled water (400 ml) and cold brine solution, the mixture was allowed to settle and the aqueous layer was again discarded.

[0226] b) Glu(OtBu)-Gly-OH (66.89 g), triethylamine (39.06 g) and water are added to the organic layer at 10-20° C., and the mixture is stirred at 25-30° C. for 4-6 hours. The solvent is removed under vacuum at 40-45° C., and the reaction mixture is degassed and then cooled to 10-15° C. Precooled methanol (400 ml) and cold water (800 ml) are added to the residue, and the temperature is adjusted to 10-20° C. The pH of the reaction mixture is slowly adjusted to 3.0 to 3.5. The reaction mixture is stirred for 2-4 hours and the resulting solid is filtered, washed with cold water, and dried under vacuum for 1-2 hours.

[0227] Methanol (400 ml) was added to the obtained solid (Fmoc-Ala-Glu(OtBu)-Gly-OH) at 20-30° C., and the temperature was raised to 40-50° C., after which the reaction mixture was stirred for 1-2 hours. The reaction mixture was cooled to 20-30° C. and stirred for 1-2 hours, after which the obtained solid was filtered. The solid was washed with methanol and dried under vacuum.

[0228] Methanol (400 ml) was added to the solid obtained above, and the temperature of the reaction mixture was raised to 40-50° C., then cooled to 20-30° C. and stirred for 1-2 hours. The obtained solid was filtered, washed with methanol, and dried under vacuum for 1-2 hours.

[0229] c) Dichloromethane (400ml) and diethylamine (80ml) are added to the solid at 20-30°C, and the reaction mixture is stirred for 7-9 hours. Distilled water (400ml) is added to the reaction mixture and stirred for 20-25 minutes, then allowed to stand. The layers are separated. The aqueous layer is washed with dichloromethane and then distilled under vacuum at a temperature below 50°C. Dichloromethane (400ml) is added thereto and then recovered under vacuum at a temperature below 40°C. Dichloromethane (400ml) is added again and the residue is heated to 30-40°C. Diisopropyl ether (400ml) is added, the reactants are stirred for 60 minutes, cooled to 20-30°C, and further stirred for 2-3 hours. The solid (46g) is isolated by filtration, washing with diisopropyl ether and drying under vacuum.

[0230] Yield: 50.94% (purity 98.3%).

[0231] Stage-3: Preparation of tetrapeptide: (Boc)-His(Trt)-Ala-Glu(OtBu)-Gly-OH)

[0232]

[0233] a) EDC-HCl (34.52 g) is added to a mixture of HONB (32.25 g) and Boc-His(Trt)-OH (60 g) in tetrahydrofuran (480 ml). The mixture is stirred at 25-30°C for 12-15 hours and cooled to 10-15°C. Precooled distilled water (300 ml) and cold brine solution are simultaneously added to the reaction mixture and stirred. The mixture is allowed to stand and the layers are separated, and the aqueous layer is discarded.

[0234] The organic layer was washed with pre-cooled distilled water (300 ml) and cold brine solution. The layers were separated.

[0235] 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. The reaction mixture was stirred at 25-30°C for 2-4 hours and then cooled to 10-15°C. Precooled distilled water (300 ml) and precooled dichloromethane (600 ml) were added, and the pH was adjusted to 3 to 3.5. The mixture was stirred and allowed to stand to separate the layers. The organic layer was washed with precooled sodium bicarbonate solution and precooled saline solution at 10-15°C. The aqueous layer was discarded.

[0236] 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 and degassed under vacuum. Ethyl acetate (600 ml) was added to the obtained residue, and the temperature was raised to 40-45° C., after which the material was cooled to 20-30° C., stirred, and filtered to obtain a solid. The solid was washed with ethyl acetate and dried under vacuum.

[0237] Dichloromethane (2400 ml) was added to the solid and the reaction mixture was heated to 33-35°C and stirred to obtain a clear solution. Distilled water (600 ml) and brine solution were added and the reaction mixture was stirred and allowed to stand to separate the layers.

[0238] The organic layer was washed with distilled water and brine solution, recovered under vacuum at a temperature below 50°C and degassed to obtain a residue.

[0239] Ethyl acetate (600 ml) was added to the residue. The temperature was raised to 40-45°C. The reaction was cooled to 20-30°C, stirred, and filtered to obtain a solid. The solid (63 g) was washed with ethyl acetate (60 ml) and dried under vacuum.

[0240] 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 R1 and R2 are independently selected from amine protecting groups, R3 is t-Bu or Bn, and A is an acid activating group.

2. The method according to claim 1, wherein R1 is selected from the group consisting of tert-butyloxycarbonyl, trityl, 4-methyltrityl, monomethoxytrityl, carboxybenzyl, and fluorenylmethoxycarbonyl; and R2 is selected from the group consisting of 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 R4 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 the group consisting of 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 of 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.

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