DESC:

FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10; rule 13)
1. TITLE OF THE INVENTION–PROCESS FOR THE PREPARATION OF TETRAPEPTIDE FRAGMENT OF LIRAGLUTIDE
2. Applicant(s)
A) NAME: FRESENIUS KABI ONCOLOGY LTD.
B) NATIONALITY: An Indian Company
C) ADDRESS: B-310, SOM DATT CHAMBERS-I, BHIKAJI CAMA PLACE, NEW DELHI -110066, INDIA
3. PREAMBLE OF THE DESCRIPTION
The following specification particularly describes the invention and the manner in which it is to be performed.

FIELD OF THE INVENTION
The present invention relates to an improved process for the preparation of a tetrapeptide of formula I, used in the synthesis of Liraglutide.

Formula I
wherein R1 and R2 are independently selected from an amine protecting group, and R3 is t-Bu or Bn.
The present invention further, relates to a process for the preparation of Liraglutide or its pharmaceutically acceptable salts, using the tetrapeptide of formula I prepared by the process of the present invention.
BACKGROUND OF THE INVENTION
Liraglutide, a human GLP-1 receptor agonist (or GLP-1 analogue) is represented by the formula,

Liraglutide, sold under the brand name Victoza ® (Novo Nordisk), is an anti-diabetic medication used to treat type 2 diabetes, obesity, and chronic weight management. It was approved for medical use in the European Union in 2009, and in the United States in 2010.
Liraglutide and its preparation are disclosed in US6268343.
Generally, Liraglutide is prepared by chemical synthesis, either via solid phase synthesis or liquid phase by sequential coupling of amino acids, or by a convergent synthesis involving coupling of fragments, which were synthesised separately.
Most of the processes known in the art for synthesis of Liraglutide involve synthesis of various short chain peptide fragments such as a dipeptide, a tripeptide, a tetrapeptide, a pentapeptide and the like, either in solution or solid phase. These fragments are then coupled either in solid or solution phase or via a hybrid approach, to provide Liraglutide.
The tetrapeptide fragment of formula I is one such fragment, which is used in the synthesis of Liraglutide.
Various processes for the synthesis of the tetrapeptide fragment of formula I with suitable protecting groups in solid as well as liquid phase, are known in the art.
WO2016046753A1 discloses a process for the synthesis of the tetrapeptide fragment where it is prepared by a solid phase synthesis with a purity of 94% by HPLC.
A solid phase synthesis of tetrapeptide of formula I is also disclosed in CN102875665B and CN106478805B with a purity of about 94% and 96.6% by HPLC respectively.
However, the solid phase synthesis of short chain peptides like tetrapeptide of formula I is not economically and commercially viable due to the use of large volumes of solvents during the process. The recovery of final product is also cumbersome due to numerous resin washings, elution, and purification of the product by large volume of solvents. There is also a limitation on scale up of batch sizes. Therefore, synthesis of short chain peptide fragments via solid phase is generally, not favoured, as the process is not worth the efforts involved in the synthesis.
CN105732798B discloses another process for the synthesis of the tetrapeptide fragment of Formula I. The disclosed process involves a linear coupling of histidine (His) with alanine (Ala), glutamic acid (Glu) and glycine (Gly) in presence of a solvent and a base.
The tetrapeptide of formula I contains histidine at the amino terminal. Histidine is an amino acid that is highly prone to racemization. On exposure to base, the tendency of histidine towards racemization increases.
The process as disclosed in CN105732798B results in exposure of histidine amino acid to a base during two stages of synthesis leading to racemization of histidine and formation of D-isomeric impurity of histidine in the tetrapeptide which is carried further to the final API stage, when the tetrapeptide with high D-isomeric histidine impurity is converted to Liraglutide. Separation of this impurity from the final peptide is extremely difficult and thus the final peptide can contain varying amounts of D-His impurity. Thus, Liraglutide product with high level of D-isomeric impurity is obtained which, therefore, is unsuitable for pharmaceutical preparations.
From the foregoing, it is apparent that the reported methods for the preparation of tetrapeptide fragment of Liraglutide, require complex operational conditions and do not provide highly pure product, especially with respect to D-isomeric histidine impurity.
The processes disclosed in various prior arts, also involve tedious work up procedures and various purification steps and are also not cost effective.
Thus, there remains a need to formulate an efficient, simple, and industrially viable synthetic process which can overcome the drawbacks of the prior art and provide tetrapeptide fragment of formula I as well as Liraglutide in high purity in a cost-effective manner.
OBJECT OF THE INVENTION
It is an object of the present invention to overcome the above-mentioned drawbacks of the prior art.
It is another objective of the present invention to provide an improved and commercially viable process for the synthesis of the tetrapeptide fragment of formula I and henceforth, for the synthesis of Liraglutide using the tetrapeptide fragment of formula I prepared by the process of the present invention.
It is a further objective of the present invention to obtain tetrapeptide fragment of formula I and Liraglutide or its pharmaceutically acceptable salts thereof, in high purity.
SUMMARY OF THE INVENTION
The present invention relates to an improved process for the preparation of tetrapeptide of formula I, used in the synthesis of Liraglutide.
In an aspect, the present invention relates to an improved process for the preparation of a tetrapeptide of formula I,

Formula I
wherein R1 and R2 are independently selected from an amine protecting group, and R3 is t-Bu or Bn,
comprising the step of condensing an activated compound of formula II,

Formula II
wherein R1 and R2 are as defined above; and A is an acid activating group.
with a tripeptide of formula III,

Formula III
wherein R3 is as defined above.
In a further aspect, the present invention relates to a process for the preparation of Liraglutide or its pharmaceutically acceptable salts thereof, comprising converting the tetrapeptide of formula I, obtained by the process of the present invention, to Liraglutide or its pharmaceutically acceptable salts thereof.
DEFINITIONS
The following definitions are used in connection with the present application unless the context indicates otherwise.
“Peptide” refers to a short chain of amino acids, wherein two or more amino acids are chemically linked by an amide linkage.
“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 of two or more amino acids present. The amino acids in the fragment may be protected or unprotected.
The term “condensing” refers to a condensation reaction where an amide linkage is formed by reaction of a carboxylic group with an amine group.
The term “carboxylic acid” refers to an organic compound that contains a -COOH group.
The term “amine” refers to an organic compound that contains a -NH2 group.
The term “activated compound” refers to a compound with an activated ester group.
The term “acid activating group” refers to a group that enhances the reactivity of a carboxylic group. An activated carboxylic acid undergoes the same reactions as their inactivated analogues but do so more rapidly.
The term “activated ester” refers to an ester functional group that is highly susceptible towards nucleophilic attack. An activated ester undergoes the same reactions as their inactivated analogues but do so more rapidly. An activated ester can be prepared by conversion of the hydroxyl (–OH) of the carboxylic acid group into a good leaving group by reaction with an activating agent.
The term “leaving group” refers to an atom, a group of atoms or to a fragment that detaches from the main or residual part of a substrate during a reaction or elementary step of a reaction.
The term “activating agent” refers to a compound that causes activation of a molecule or a group towards reaction with another group or a molecule by donating an activating group into the molecule.
The term “coupling reagent” refers to a reagent that facilitates formation of a bond between two adjacent groups.
The term “protecting group” refers to a temporarily attached group to a functional group to decrease the reactivity of the functional group so that the protected functional group does not react under synthetic conditions to which the molecule is subjected in one or more subsequent steps, at the same time allowing for removal of such protecting group under conditions, which do not harm the remaining molecule.
The term “amine protecting group” specifically refers to a protecting group that is attached to an amine functionality in a molecule.
For the purpose of the present invention, an “amine protecting group” in a molecule having two or more amine protecting groups, may be the same or different from one another.
The protecting groups can be cleaved from the molecule after the desired compound is obtained. The term “deprotecting” refers to the cleavage or removal of the protecting groups.
The protecting group can be deprotected under acidic, basic and/or neutral conditions. The protection and deprotection methods are well known in the art (see notably "Protective groups in organic synthesis", Greene T. W. and Wuts P. G. M., Wiley-Interscience, 1999).
The term “ambient temperature” refers to a temperature ranging from about 15°C to 35°C.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a process for the preparation of a tetrapeptide of formula I,

Formula I
wherein R1 and R2 are independently selected from an amine protecting group, R3 is t-Bu or Bn.
In some embodiments, the amine protecting group is selected from the group consisting of tert-butyloxycarbonyl, trityl, 4-methyltrityl, monomethoxytrityl, fluorenylmethyloxycarbonyl, carboxybenzyl, N-benzyloxymethyl and toluenesulfonyl.
In one embodiment R1 and R2 are the same amine protecting groups.
In another embodiment, R1 and R2 are different amine protecting groups.
In a preferred embodiment, R1 is selected from tert-butyloxycarbonyl, trityl, 4-methyltrityl, monomethoxytrityl, carboxybenzyl, fluorenylmethyloxycarbonyl.
In another preferred embodiment, R2 is selected from trityl, 4-methyltrityl, monomethoxytrityl, N-benzyloxymethyl, fluorenylmethyloxycarbonyl, toluenesulfonyl and tert-butyloxycarbonyl.
In a more preferred embodiment, R1 is tert-butyloxycarbonyl and R2 is selected from trityl and 4-methyltrityl.
In a yet more preferred embodiment, R1 is tert-butyloxycarbonyl and R2 is trityl.
In an aspect, the process comprises a step of condensing an activated compound of formula II,

Formula II
wherein R1 and R2 are as defined above; A is an acid activating group,
with a tripeptide of formula III,

Formula III
wherein R3 is as defined above.
In an embodiment, the acid activating group A in compound of formula II is the ester group introduced from the activating agents selected from 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, ethyl 1-hydroxy-1H-1,2,3-triazole-4-carboxylate, and N-hydroxytetrazole.
In an embodiment, the condensation reaction of the compound of formula II and the compound of formula III is carried out in presence of a base and a solvent.
The base can be selected from the group consisting of N, N-diisopropylethylamine, triethylamine, methyl morpholine, sodium bicarbonate, sodium carbonate and potassium carbonate, preferably triethylamine.
The solvent can be selected from the group consisting of dichloromethane, 1-methyl-pyrrolidin-2-one, acetonitrile, tetrahydrofuran, dimethyl sulfoxide, N, N-dimethyl formamide, N-methyl-formamide, dimethyl carbonate, diethyl carbonate and a mixture thereof. Preferably, the solvent is dichloromethane.
In yet another embodiment, the condensation reaction is carried out at an ambient temperature, preferably at a temperature of 20°C to 35°C, more preferably at a temperature of 20°C to 30°C.
In a preferred embodiment, the tetrapeptide of formula I is a compound of Formula Ia [Formula I, wherein R1 is Boc, R2 is Trt, R3 is t-Bu].

Formula Ia
In a preferred embodiment, the present invention relates to a process of preparation of a compound of formula Ia comprising condensing an activated compound of formula IIa [Formula II, wherein R1 is Boc and R2 is Trt and A is ONB],

Formula IIa
with a tripeptide of formula IIIa [Formula III, wherein R3 is t-Bu]

Formula IIIa.
In yet another preferred embodiment, condensation is carried out in presence of dichloromethane and triethylamine at a temperature of 25°C to 30°C . The reaction mixture is stirred for 2-4 hours and the tetrapeptide of formula Ia is 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 carboxylic group of a compound of formula IV,

Formula IV
wherein R1 and R2 are as defined above
with an activating agent.
The activating agent can 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, ethyl 1-hydroxy-1H-1,2,3-triazole-4-carboxylate and N-hydroxytetrazole. Preferably, the activating agent is N-hydroxy-5-norbornene-2,3-dicarboximide.
In an embodiment, the activation of the carboxylic group is carried out in the presence of a coupling reagent in a solvent.
The coupling reagent can be selected from the group consisting of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, N, N-dicyclohexylcarbodiimide, Oxyma B /diisopropyl carbodiimide, benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate, hexafluorophosphate azabenzotriazole tetramethyl uronium, O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate and propylphosphonic anhydride. Preferably, the coupling agent is selected from the group consisting of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, N, N-dicyclohexylcarbodiimide and Oxyma B /diisopropyl carbodiimide. More preferably, the coupling agent is N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride.
The solvent can be selected from the group consisting of dichloromethane, 1-methyl-pyrrolidin-2-one, acetonitrile, tetrahydrofuran, dimethyl sulfoxide, N, N-dimethyl formamide, N-methyl-formamide, dimethyl carbonate, diethyl carbonate and a mixture thereof. Preferably, the activation of the carboxylic group with an activating agent is carried out in the presence of a coupling reagent in tetrahydrofuran.
In a preferred embodiment, the compound of Formula IV, wherein R1 is Boc and R2 is Trt, is activated with N-hydroxy-5-norbornene-2,3-dicarboximide (HONB) to form activated compound of formula IIa[Formula II, wherein R1 is Boc and R2 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 (HONB) in tetrahydrofuran in presence of N-(3-dimethylaminopropyl)-N'-ethyl carbodiimide hydrochloride at 20-30°C temperature for 10 to 12 hours. The activated compound of formula II is isolated after the completion of reaction by addition of water and dichloromethane.
In an embodiment, the present invention relates to process for the preparation of a tripeptide of formula III comprising a step of activating the carboxylic group of a compound of formula V,

Formula V
wherein R4 is an amine protecting group,
with an activating agent in presence of a coupling reagent in a solvent to obtain an activated compound of formula VI,

Formula VI
wherein R4 and A is defined as above.
The amine protecting group (R4) can be selected from fluorenylmethyloxycarbonyl, tert-butyloxycarbonyl, carboxybenzyl and toluenesulfonyl, preferably it is fluorenylmethyloxycarbonyl.
The activating agent can 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, ethyl 1-hydroxy-1H-1,2,3-triazole-4-carboxylate and N-hydroxytetrazole. Preferably, the activating agent is N-hydroxy-5-norbornene-2,3-dicarboximide.
In an embodiment, the activation of the carboxylic group is carried out in the presence of a coupling reagent in a solvent.
The coupling reagent can be selected from the group consisting of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, N, N-dicyclohexylcarbodiimide, Oxyma B /diisopropyl carbodiimide, benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate, hexafluorophosphate azabenzotriazole tetramethyl uronium, O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate and propylphosphonic anhydride. Preferably, the coupling agent is selected from the group consisting of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, N, N-dicyclohexylcarbodiimide and Oxyma B /diisopropyl carbodiimide. More preferably, the coupling agent is N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride.
The solvent can be selected from the group consisting of dichloromethane, 1-methyl-pyrrolidin-2-one, acetonitrile, tetrahydrofuran, dimethyl sulfoxide, N, N-dimethyl formamide, N-methyl-formamide, dimethyl carbonate, diethyl carbonate and a mixture thereof. Preferably, the activation of the carboxylic group with an activating agent is carried out in the presence of a coupling reagent in tetrahydrofuran.
In a preferred embodiment, the compound of formula V, wherein R4 is Fmoc, is activated with N-hydroxy-5-norbornene-2,3-dicarboximide (HONB) to give activated compound of formula VI , wherein R4 is Fmoc; A is ONB.
In another preferred embodiment, the compound of formula V is treated with N-hydroxy-5-norbornene-2,3-dicarboximide (HONB) in tetrahydrofuran in presence of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride at 25°C to 30°C for 10 to 12 hours to give the activated compound of formula VI. The activated compound of formula VI is isolated in dichloromethane after the completion of reaction by addition of water and dichloromethane.
In an embodiment, the activated compound of formula VI is further condensed with a dipeptide of formula VII,

Formula VII
wherein R3 is t-Bu or Bn
in presence of a base in a solvent to obtain a protected tripeptide of formula VIII,

Formula VIII
wherein R3 is t-Bu or Bn and R4 is an amine protecting group.
The amine protecting group (R4) can be selected from fluorenylmethyloxycarbonyl, tert-butyloxycarbonyl, carboxybenzyl and toluenesulfonyl, preferably it is fluorenylmethyloxycarbonyl.
The base can be selected from the group consisting of N, N-diisopropylethylamine, triethylamine, methyl morpholine, sodium bicarbonate, sodium carbonate and potassium carbonate. Preferably the base is triethylamine.
The solvent can be selected from the group consisting of dichloromethane, 1-methyl-pyrrolidin-2-one, acetonitrile, tetrahydrofuran, dimethyl sulfoxide, N, N-dimethyl formamide, N-methyl-formamide, dimethyl carbonate, diethyl carbonate and a mixture thereof. Preferably, the solvent is dichloromethane.
In a preferred embodiment, the activated compound of formula VI, wherein R4 is Fmoc; A is ONB, is condensed with the dipeptide of formula VII, wherein R3 is t-Bu, to provide a protected tripeptide of formula VIII, wherein R3 is t-Bu, R4 is Fmoc.
In another preferred embodiment, condensation is carried out in the presence of triethylamine and dichloromethane as solvent.
The tripeptide of formula III is obtained by deprotecting the protected tripeptide of formula VIII by any of the known methods in the art. The deprotection is performed either in an acid or a base, depending on the protecting group to be removed.
Preferably, the protected tripeptide of formula VIII is deprotected in presence of an organic base in a solvent.
The organic base can be selected from the group consisting of ammonia, diethylamine, piperidine, piperazine, tributylamine, pyrrolidine, ethanolamine, morpholine, 1,8-diazabicyclo [5.4.0] undec-7-ene, 1,4-diazabicyclo [2.2. 2]octane, and dicyclohexylamine. Preferably, the organic base is diethylamine.
The solvent in the deprotection reaction can be selected from the group consisting of dichloromethane, 1-methyl-pyrrolidin-2-one, acetonitrile, tetrahydrofuran, dimethyl sulfoxide, N, N-dimethyl formamide, N-methyl-formamide, dimethyl carbonate, diethyl carbonate and a mixture thereof. Preferably, this solvent is dichloromethane.
More preferably, the deprotection of the protected tripeptide of Formula VIII can be carried out in the presence diethylamine in dichloromethane solvent.
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 some embodiments, the solid is isolated by additions of diisopropyl ether, filtration and drying.
The dipeptide of formula VII is obtained by a process comprising a step of activating the carboxylic group of a compound of formula IX,

Formula IX
wherein R5 is an amine protecting group,
with an activating agent to provide a compound of formula X,

. Formula X
Wherein R5 and A are as defined above,
The amine protecting group R5 can be selected from the group consisting of fluorenylmethyloxycarbonyl, tert-butyloxycarbonyl, carboxybenzyl and toluenesulfonyl; preferably it is fluorenylmethyloxycarbonyl.
The activation of formula IX is done by an activating agent in presence of a coupling reagent and a solvent.
The activating agent can be selected from the group consisting of N-hydroxysuccinimide, N-hydroxy-5-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, ethyl 1-hydroxy-1H-1,2,3-triazole-4-carboxylate and N-hydroxytetrazole. Preferably, the activating agent is N-hydroxy-5-norbornene-2,3-dicarboximide.
In an embodiment, the activation of the carboxylic group is carried out in the presence of a coupling reagent in a solvent.
The coupling reagent can be selected from the group consisting of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, N, N-dicyclohexylcarbodiimide, Oxyma B /diisopropyl carbodiimide, benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate, hexafluorophosphate azabenzotriazole tetramethyl uronium, O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate and propylphosphonic anhydride. Preferably, the coupling agent is selected from the group consisting of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, N, N-dicyclohexylcarbodiimide and Oxyma B /diisopropyl carbodiimide. More preferably, the coupling agent is N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride.
The solvent can be selected from the group consisting of dichloromethane, 1-methyl-pyrrolidin-2-one, acetonitrile, tetrahydrofuran, dimethyl sulfoxide, N, N-dimethyl formamide, N-methyl-formamide, dimethyl carbonate, diethyl carbonate and a mixture thereof. Preferably, the activation of the carboxylic group with an activating agent is carried out in the presence of a coupling reagent in tetrahydrofuran.
In a preferred embodiment, the compound of formula IX, wherein R5 is Fmoc, is activated with N-hydroxy-5-norbornene-2,3-dicarboximide (HONB) to give activated compound of formula X, wherein R5 is Fmoc; A is ONB.
In a preferred embodiment, the compound of formula IX is treated with N-hydroxy-5-norbornene-2,3-dicarboximide (HONB) in tetrahydrofuran in presence of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride at 20-30°C for 6 to 8 hours, resulting in the activation of the compound of formula IX. The compound of formula X, so obtained, is extracted in dichloromethane after the completion of reaction by addition of water and dichloromethane.
Starting compound of formula IX wherein R5 is Fmoc, may be obtained from commercially available sources.
The activated compound of formula X is then condensed with Glycine (Gly) to provide a protected dipeptide which after deprotection yields the dipeptide of formula VII.
In a preferred embodiment, the condensation of the compound of formula X with Glycine is done in presence of a solvent, preferably dichloromethane and a base, preferably triethylamine.
The dipeptide of formula VII is obtained by deprotecting the protected dipeptide obtained above by any of the known methods in the art. The deprotection is performed either in an acid or a base, depending on the protecting group to be removed.
Preferably, the protected dipeptide is reacted with an organic base in a solvent.
The organic base is selected from the group consisting of ammonia, diethylamine, piperidine, piperazine, tributylamine, pyrrolidine, ethanolamine, morpholine, 1,8-diazabicyclo [5.4.0] undec-7-ene, 1,4-diazabicyclo [2.2. 2]octane, and dicyclohexylamine. Preferably the organic base is diethylamine.
The solvent used for the deprotection reaction in the presence of organic base, can be selected from the group consisting of dichloromethane, 1-methyl-pyrrolidin-2-one, acetonitrile, tetrahydrofuran, dimethyl sulfoxide, N, N-dimethyl formamide, N-methyl-formamide, dimethyl carbonate, diethyl carbonate and a mixture thereof. Preferably, the solvent is dichloromethane.
More preferably, the deprotection is carried out in the presence of diethylamine in 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 addition of water and dichloromethane followed by filtration and drying to yield the dipeptide of formula VII.
In some embodiments, the activated ester compounds of formula II, VI and X obtained in the process of present invention are prepared, in-situ and are not isolated during the process.
In the process of present invention, the tetrapeptide of formula I is prepared by using histidine in the last stage of the reaction sequence. This restricts the exposure of histidine to the base during the process. Using the process of the present application, the exposure of the histidine residue/histidine containing peptide to the base occurs only once during the synthesis and hence, racemization of histidine to D-isomer is minimised.
Surprisingly, the inventors have found that by following the sequence of reaction steps and performing the condensation of amino acids with fragments in the order according to the process of the present application, e.g., introducing histidine at the last stage, the impurity of D-isomer of histidine in the tetrapeptide fragment is highly reduced.
The prior art process instead discloses introducing histidine at the first stage, thereby exposing histidine residue to base, at every stage during subsequent reaction steps until the formation of the tetrapeptide is completed. This results in racemisation of histidine during the process and thereby yields the product with a high amount of impurity, specifically with a high amount of D-histidine impurity.
The inventors of the present invention have also tried the synthesis of tetrapeptide of formula I by introducing histidine at second stage instead of the first stage to reduce the exposure of histidine to base during the process. This also resulted in increased racemisation of histidine leading to D-His impurity of about 3%, whereas the process of the present invention yields the tetrapeptide fragment with negligible amount of D-isomer of histidine, without any further purification. The tetrapeptide of formula I obtained by the process of present invention contains less than 0.5% of D-isomeric impurity of histidine by HPLC, preferably less than 0.4% of D-isomeric impurity of histidine by HPLC, more preferably less than 0.3% of D-isomeric impurity of histidine by HPLC, and even more preferably less than 0.2% of D-isomeric impurity of histidine by HPLC.
The process according to the invention results in a tetrapeptide of formula I with a very low amount of D-isomer of histidine, impurity, (e.g., less than 0.5% by HPLC) even in the crude tetrapeptide. Further, crude product may be purified using processes known in the art to remove unreacted compounds (e.g., amino acids). However, according to the invention, the purification of crude tetrapeptide is carried out to remove the unreacted starting amino acids only and is not required for removing D-His impurity which is already controlled in the crude product obtained by employing the process of the present invention.
In some embodiments, the tetrapeptide of formula I is purified with a solvent selected from the group consisting of ethyl acetate, acetone, isopropyl alcohol, tetrahydrofuran, acetonitrile and mixtures thereof.
The tetrapeptide of formula I obtained by the process of according to the invention has a purity of 98% by HPLC or more, preferably 99% by HPLC or more, most preferably 99.4% by HPLC.
Preferably, the process according to the invention is carried out in solution phase. This also eliminates the disadvantages of solid phase synthesis such as use of costly resins, multiple washing with solvents, and problems of scale up of batches.
In a further embodiment, the tetrapeptide of formula I, obtained by a process of the present invention may be converted to Liraglutide or its pharmaceutically acceptable salts by the methods known in the art, for example, using a process as reported in WO2016046753A1.
The use of a highly purified tetrapeptide fragment of formula I as obtained by the process of the present invention, positively impacts the yield and purity of the Liraglutide peptide or its salts.
Thus, the invention provides liraglutide or its pharmaceutically acceptable salt thereof which contains less than 0.5% of D-isomeric impurity of histidine by HPLC, preferably less than 0.4% of D-isomeric impurity of histidine by HPLC, more preferably less than 0.3% of D-isomeric impurity of histidine by HPLC, and even more preferably less than 0.2% of D-isomeric impurity of histidine by HPLC.
Thus, the inventors of the present invention have developed an improved process, not only for the synthesis of the tetrapeptide fragment but also for Liraglutide or its pharmaceutically acceptable salts, which is both cost efficient and commercially viable.
The process can be scaled up effectively and the product or its intermediates at various stages of synthesis can be isolated using isolation techniques such as solvent extraction and solvent recovery, precipitation, distillation, filtration and drying of the product.
The synthetic method of the present invention thus, improves peptide purity, reduces material and purification cost and is advantageous for industrial production.
ABBREVIATIONS/ACRONYMS:
HONB: N-hydroxy-5-norbornene-2,3-dicarboximide, accordingly ONB refers to the same molecule when attached to a second molecule via the hydroxy group, therefore without H at the hydroxy group.
EDC. HCl: N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride
DCC: Dicyclohexyl carbodiimide
Boc: Tert-butyloxycarbonyl
Fmoc: Fluorenylmethyloxycarbonyl
Trt: Trityl
Bn-: Benzyl-
His: Histidine
Ala: Alanine
Glu: Glutamic Acid
Gly: Glycine
t-Bu: t-Butyl
OH: Hydroxyl
D-Isomer: Dextrorotatory isomer
HPLC: High Performance Liquid Chromatography

EXPERIMENTAL
Detailed experimental parameters according to 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 example
Preparation of tetrapeptide: (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°C for 15-16 hours. Distilled water (750 ml) and brine solution (150 ml) was added into the reaction mixture and stirred for 190 minutes and allowed to settle. The aqueous layer was separated and discarded. Water (750 ml) and brine solution (150 ml) was added to the organic layer. The mixture was stirred. The layers were separated, and the aqueous layer was discarded.
b) To the organic layer, triethylamine solution (35.7 g in tetrahydrofuran 150 ml) and glycine solution (26.5 g dissolve in 300 ml distilled water) was added slowly at 20-30°C and the reaction mixture was stirred for 3 to 5 hours. Distilled water (600 ml) and ethyl acetate (600 ml) was added into the reaction mixture and pH was adjusted between 3 to 4. The reaction mass was stirred for 10 minutes at 20-30°C. The layers were allowed to settle and separated. Sodium bicarbonate solution (52.5 g in 750 ml distilled water) and brine solution (150 ml) was added to the organic layer and the mixture was stirred for 15 minutes at 20-30°C, settled and the layers were separated. Distilled water was added to the organic layer, and pH of the reaction mass was adjusted between 3 to 4. The reaction mass was stirred for 10 minutes, settled and the layers were separated. The aqueous layer was discarded, and the organic layer was recovered under vacuum at a temperature below 40 °C. The residual mass was degassed for 120 minutes under vacuum and dichloromethane (300 ml) was added to the reaction mass. The mixture was stirred to get a clear solution and was heated to 35 to 40°C. The solid was isolated from the reaction mixture by addition of diisopropylether, cooling, filtration and drying under vacuum.
c) Dichloromethane (450 ml) and Fmoc-Glu (Ot-Bu)-Gly (90 gm) was stirred at 20-30 °C for 5-10 mins. 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 into the reaction mass and stirred for 10 min. The mixture was settled, and the layers were separated. Dichloromethane (450 ml) was added to 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. To the residual mass, dichloromethane (300 ml) was added and was recovered under vacuum at a temperature below 45°C. The residual mass was degassed under vacuum for 1 to 2 hours at 35 to-45°C. The solid (44.5g) was isolated by addition of dichloromethane followed by filtration, washing with dichloromethane, and drying under vacuum at a temperature 40-50°C for 14-16 hours.
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°C until completion of reaction. The reaction mass was cooled to 0-10°C and L-alanine solution (9 g in 125 ml demineralised water) and a solution of Triethylamine (20.4 gm in 50 ml acetonitrile) was added into it. Distilled water (1000ml) and Ethyl acetate (500 ml) was added to the reaction mass. 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 1000 ml water). The layers were separated, and the aqueous layer was discarded. Distilled water was added to the organic layer, and the pH was adjusted to 3.5 with hydrochloric acid. The layers were separated, and the aqueous layer was discarded. The organic layer was washed with distilled water and distilled at 40-45°C and degassed. Acetonitrile (500 ml) was added to the resulting mass and heated up to 40-50°C for 30 min. and then cooled to 35-40 °C. Dichloromethane (50 ml) was added to the reaction mixture and stirred for 1 hour and then cooled to 20-25°C. The reaction mixture was stirred for 4 hours at 20-30°C and the solid was filtered and washed with acetonitrile (100 ml) followed by n-Hexane (100 ml). The mass was filtered and dried in vacuum to obtain a solid (42g).
Yield: 73.4%

Stage-3: Preparation of tetrapeptide: (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 for overnight at 20-30°C until completion of reaction. The reaction mass was cooled to 0-5°C, filtered and washed with tetrahydrofuran (20ml). Glu(OtBu)-Gly-OH (5.5gm) in tetrahydrofuran (10 ml) was added into the reaction mass at 0-10 °C. Triethylamine (5 ml) was added and the reaction mass was stirred at 20-30°C for 2 hours until reaction was completed. The reaction mass was evaporated under vacuum at 35-40°C and the obtained residue was diluted with Ethyl Acetate (150 ml). Water was added to the diluted mass and the pH was adjusted to 3.4-3.5 with hydrochloric acid solution. The organic layer was separated and washed with saturated NaHCO3 solution (100ml). Water was added to the organic layer the pH was adjusted to 4.0-4.1 with hydrochloric acid solution. The organic layer was separated and evaporated under vacuum at 35-40°C. Water and dichloromethane (50 ml) was added to the reaction mass and stirred. The dichloromethane layer was separated and slowly added to diisopropyl ether (150 ml). The precipitated mass was filtered, washed with di isopropyl ether and dried under vacuum at to obtain a solid (6g).
Yield: 42.07% (Purity: 85.1%); D-isomer: 2.99%.
Example-1
Preparation of tetrapeptide: (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°C for 15-16 hours. Distilled water (750 ml) and brine solution (150 ml) was added into the reaction mixture and stirred for 190 minutes and allowed to settle. The aqueous layer was separated and discarded. Water (750 ml) and brine solution (150 ml) was added to the organic layer. The mixture was stirred. The layers were separated, and the aqueous layer was discarded.
b) To the organic layer, triethylamine solution (35.7 g in tetrahydrofuran 150 ml) and glycine solution (26.5 g dissolve in 300 ml distilled water) was added slowly at 20-30°C and the reaction mixture was stirred for 3 to 5 hours. Distilled water (600 ml) and ethyl acetate (600 ml) was added into the reaction mixture and pH was adjusted between 3 to 4. The reaction mass was stirred for 10 minutes at 20-30°C. The layers were allowed to settle and separated. Sodium bicarbonate solution (52.5 g in 750 ml distilled water) and brine solution (150 ml) was added to the organic layer and the mixture was stirred for 15 minutes at 20-30°C, settled and the layers were separated. Distilled water was added to organic layer, and pH of the reaction mass was adjusted between 3 to 4. The reaction mass was stirred for 10 minutes, settled and the layers were separated. The aqueous layer was discarded, and the organic layer was recovered under vacuum at a temperature below 40 °C. The residual mass was degassed for 120 minutes under vacuum and dichloromethane (300 ml) was charged into the mass. The mixture was stirred to get a clear solution and was heated to 35 to 40°C. The solid was isolated from the reaction mixture by addition of diisopropylether, cooling, filtration and drying under vacuum.
c) Dichloromethane (450 ml) and Fmoc-Glu(OtBu)-Gly (90 gm) was stirred at 20-30 °C for 5-10 mins. 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 into the reaction mass and stirred for 10 min. The mixture was settled, and the layers were separated. Dichloromethane (450 ml) was added to aqueous layer and stirred for 10-15 minutes. The layers were again separated. The aqueous layer was distilled under vacuum at a temperature below 55°C. To the residual mass, dichloromethane (300 ml) was charged and was recovered under vacuum at a temperature below 45°C. The residual mass was degassed under vacuum for 1 to 2 hours at 35 to-45°C. The solid (44.5g) was isolated by addition of dichloromethane followed by filtration, washing with dichloromethane and drying under vacuum at a temperature 40-50°C for 14-16 hours.
Yield: 48.5% (Purity: 98.2%).
Stage -2: Preparation of Tripeptide: Ala-Glu (OtBu)-Gly-OH

a) EDC. HCl ((92.40 g) was added to a mixture of HONB (86.32g) and Fmoc-Ala (100 g) in tetrahydrofuran (500 mL) and was stirred at 20-30°C for 10 to 12 hours. After completion of the reaction, distilled water and dichloromethane (500 ml) was added into the reaction mixture and stirred for 10-15 minutes and allowed to settle. The aqueous layer was separated, and the product was extracted in dichloromethane solvent. The dichloromethane layers were combined, and water was added. The layers were separated, and the aqueous layer was discarded. The organic layer was retained.
b) To the dichloromethane layer, Glu (OtBu)-Gly-OH (83.59 g) was added at an 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 was stirred for 2 to 4 hours. The solid was isolated from the reaction mixture by filtration, washings with water and extractions with methanol at a temperature of 35 to 45°C and cooling to 20-30°C.c) Dichloromethane (500 ml) and diethylamine (100 ml) was added to solid (Fmoc-Ala-Glu(OtBu)-Gly-OH) obtained at step b), at 20-30°C .The reaction mass was stirred for 5-8 hours, and distilled water was added. The layers were separated, and the aqueous layer was recovered under vacuum below 50°C. Dichloromethane (500 ml) was added to the above residue and the mixture was heated to 35-45°C. The solid (71g) was isolated from the reaction mixture by additions of diisopropyl ether, filtration and drying under vacuum at 40-50°C for 15-18 hours.
Yield: 66.7% (Purity: 94.8%).
Stage-3: Preparation of tetrapeptide: (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°C for 10 to 12 hours. After completion of the reaction, distilled water and dichloromethane (500 ml) was added to the reaction mixture and stirred for 10-15 minutes and allowed to settle.
b) Ala-Glu (OtBu)Gly-OH (66.57 g) was charged into the dichloromethane layer from the above step and triethylamine (30.5 g) and distilled water (50 ml) was added into it. The reaction mass was stirred for 2 to 4 hours at 20-30°C and the pH of the reaction mass 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 into it and stirred for 10-15 minutes. The reaction mixture was settled, and layers were separated. The organic layer was recovered under vacuum at a temperature below 45°C to obtain a residue (crude product) D-isomer: 0.34%).
Ethyl acetate (500 ml) was added to the residue and the temperature was raised to 35-45°C. The reaction mass was stirred for 2-3 hours at 35-45°C and cooled to 20-30°C temperature. The solid (118g) was isolated by filtration and washing with ethyl acetate and dried under vacuum for at 40-45°C for 15-18 hours.
Yield:72.4% (Purity 99.4%); D-isomer: 0.22%.

Example-2
Preparation of tetrapeptide: (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 °C for 12-15 hours. The reaction mixture was cooled to 10-15 °C. Pre-cooled distilled water (1000 mL) and cooled brine solution are added into the reaction mixture and stirred. The layers were allowed to settle, separated and the aqueous layer was discarded. The organic layer was washed with pre-cooled distilled water (1000 mL) and cooled brine solution. The mixture was again allowed to settle, and the aqueous layer was discarded.
b) To the organic layer, glycine solution and triethylamine solution was added at 10-20 °C. The reaction mixture was stirred for 4 -6 hours, and the reaction mixture was cooled to 10-15°C. Pre-cooled distilled water (800 mL) and pre-cooled ethyl acetate (2000 mL) was added to it at 10 -15 °C. The pH was adjusted to 3 to 3.5. The reaction mixture was stirred for 10-15 minutes at 10-15 °C, the layers were settled, and the aqueous layer was discarded. To the organic layer, pre-cooled sodium bicarbonate solution and cooled brine solution was added at 10-20 °C into the reaction mixture. The reaction mixture was stirred, and the layers were separated. To the organic layer, pre-cooled distilled water (1000 mL) was added at 10-15 °C. The pH was adjusted between 3 to 3.5 and the reaction mixture was stirred for 10-15 minutes. The layers were settled and separated, and the aqueous layer was discarded. The organic layer was recovered under vacuum at below 50°C temperature. Dichloromethane (400 mL) was added and then recovered under vacuum at below 50°C temperature. The residual mass was degassed under vacuum, and dichloromethane (400 mL) was added to it. Stirred to get a clear solution. The reaction mass was heated to 35 – 40 °C and diisopropylether (2000 mL) was added to and the reaction mixture was stirred for 30-60 minutes at 35 to 45 °C. The reaction mixture was cooled to 20 -30 °C and stirred for 2-3 hours and the solid obtained was filtered. The solid was washed with diisopropylether (200 mL) and dried for 100 -120 minutes under vacuum to be used as such for the next step.
To the residual mass, dichloromethane (400 mL) was added and stirred to get a clear solution. The reaction mixture was heated to 35- 40 °C and diisopropylether (200 mL) added into it. The mixture was stirred for 30-60 minutes at 35-45 °C and cooled to 20-30 °C. The mixture was stirred for 2 - 3 hours and the obtained solid was filtered, washed with diisopropylether and dried under vacuum.
c) To the residual solid, dichloromethane (1000 mL) was added, and the reaction mixture was stirred for 5- 10 minutes at 20- 30 °C. Diethylamine (200 mL) was added into it and the mixture was stirred for 8 - 10 hours. Distilled water (1000 mL) was added into 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 °C. Dichloromethane (400 mL) was added to it, and then recovered under vacuum at below 40°C temperature. To the residual mass, dichloromethane (1000 mL) was added, heated to 30-40 °C and diisopropylether (1000 mL) was then added into the reaction mass at 30-40 °C and stirred for 60 minutes. The reaction mixture was cooled to 20 to 30 °C, stirred for 2-3 hours. The solid (74g) was isolated by filtration, washing 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 was stirred at 25 to 30 °C for 12 -15 hours. The reaction mixture was cooled to 10 to15 °C. Pre-cooled distilled water (400 mL) and cooled brine solution are added into the reaction mixture and stirred.
The layers were allowed to settle, and the aqueous layer was discarded. The organic layer was washed with pre-cooled distilled water (400 mL) and cooled brine solution, the mixture was allowed to settle, and the aqueous layer was discarded again.

b) To the organic layer, Glu (OtBu)Gly-OH (66.89 g), ttriethylamine (39.06 g) and water were added at 10 -20 °C, and the mixture was stirred for 4 - 6 hours at 25-30 °C. The solvent was removed at 40-45 °C under vacuum and the reaction mixture was degassed and cooled to 10 -15 °C. Pre-cooled methanol (400 mL) and cooled water (800 mL) was added to the residual mass and the temperature was adjusted to 10-20 °C. The pH of the reaction mixture was adjusted to 3.0 to 3.5 slowly. The reaction mixture was stirred for 2-4 hours and the solid obtained was filtered, washed with cooled water, dried for 1-2 hrs under vacuum.

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 and the reaction mixture was stirred for 1-2 hours. The reaction mixture was cooled to 20-30 °C and stirred for 1-2 hours and the obtained solid was filtered. The solid was washed with methanol and dried under vacuum.

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

c) Dichloromethane (400 mL) and diethylamine (80 mL) was added to the solid at 20-30 °C and the reaction mixture was stirred for 7-9 hours. Distilled water (400 mL) was added to the reaction mixture and stirred for 20-25 minutes and settled. The layers were separated. The aqueous layer was washed with dichloromethane and then distilled under vacuum at a temperature below 50 °C. Dichloromethane (400 mL) was added to it and then recovered under vacuum at below 40 °C temperature. Dichloromethane (400 mL) was again added, and the residual mass was heated to 30-40 °C. Diisopropylether (400 mL) was added, the reaction mass was stirred for 60 minutes and cooled to 20-30 °C and further stirred for 2-3 hours. The solid (46 g) was isolated by filtration, washing with diisopropylether and drying under vacuum.
Yield: 50.94% (Purity 98.3%)
Stage-3: Preparation of tetrapeptide: (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 °C for 12- 15 hours and cooled to 10-15 °C. Pre-cooled distilled water (300 mL) and cooled brine solution was simultaneously added into the reaction mixture and stirred. The layers were settled and separated, and the aqueous layer was discarded.
The organic layer was washed with pre-cooled distilled water (300 mL) and cooled brine solution. The layers were separated.
b) Ala-Glu (OtBu)Gly -OH (39.76 g) was charged into the organic layer and triethylamine (18.21 g) and distilled water (60 mL) was added into it. The reaction mixture was stirred for 2- 4 hours at 25-30 °C and then cooled to 10-15 °C. Pre-cooled distilled water (300 mL) and pre -cooled dichloromethane (600 mL) was added into it and the pH was adjusted between 3 to 3.5 and the mixture was stirred, settled to separate the layers. The organic layer was washed with pre-cooled sodium bicarbonate solution and pre-cooled brine solution at 10-15 °C. The aqueous layer was discarded.
To the organic layer, pre-cooled distilled water (300 mL) was added, and the pH of reaction mixture was adjusted between 3 to 3.5. The mixture was stirred, settled 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 °C and the mass was cooled to 20-30 °C, stirred and filtered to obtain a solid. The solid was washed with ethyl acetate and dried under vacuum.
Dichloromethane (2400 mL) was added to the solid and the reaction mixture was heated to 33 -35 °C and stirred to get a clear solution. Distilled water (600 mL) and brine solution was added into it and the reaction mixture was stirred, settled to separate the layers.
The organic layer was washed with distilled water and brine solution, recovered under vacuum at below 50 °C temperature and degassed to obtain a residue.
Ethyl acetate (600 mL) was charged into the residue. The temperature was raised to 40-45 °C. The reaction mass was cooled to 20-30 °C, stirred and filtered to obtain a solid mass. The solid (63) was washed with ethyl acetate (60 mL) and dried under vacuum.
Yield: 64.81% (Purity 98.4%); D -isomer: 0.04%.
,CLAIMS:WE CLAIM:
1. A process for the preparation of a tetrapeptide of formula I,

Formula I

comprising the step of condensing an activated compound of formula II,

Formula II
.
with a tripeptide of formula III,

Formula III
wherein R1 and R2 are independently selected from an amine protecting group, R3 is t-Bu or Bn, and A is an acid activating group.
2. The process according to claim 1, wherein R1 is selected from tert-butyloxycarbonyl, trityl, 4-methyltrityl, monomethoxytrityl, carboxybenzyl, fluorenylmethyloxycarbonyl; and R2 is selected from trityl, 4-methyltrityl, monomethoxytrityl, N-benzyloxymethyl, fluorenylmethyloxycarbonyl, toluenesulfonyl and tert-butyloxycarbonyl.
3. The process according to claim 1, wherein the acid activating group A is an ester group introduced from an activating agent 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, ethyl 1-hydroxy-1H-1,2,3-triazole-4-carboxylate, and N-hydroxytetrazole.
4. The process according to claim 1, wherein the condensation reaction is carried out in presence of a base.
5. The process according to claim 4, wherein the base is selected from the group consisting of N, N-diisopropylethylamine, triethylamine, methyl morpholine, sodium bicarbonate, sodium carbonate and potassium carbonate.
6. The process according to claim 1, wherein the condensation reaction is carried out in presence of a solvent.
7. The process 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-dimethyl formamide, N-methyl-formamide, dimethyl carbonate, diethyl carbonate and a mixture thereof.
8. The process according to claim 1, wherein the condensation reaction is carried out at an ambient temperature.
9. The process according to claim 1, wherein the compound of formula II is prepared by activating the carboxylic group of a compound of formula IV,

Formula IV
with an activating agent in the presence of a coupling reagent in a solvent.
10. The process according to claim 9, wherein
the activating agent 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, ethyl 1-hydroxy-1H-1,2,3-triazole-4-carboxylate, and N-hydroxytetrazole,
the coupling reagent is selected from the group consisting of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, N, N-dicyclohexylcarbodiimide, Oxyma B /diisopropyl carbodiimide, benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate, hexafluorophosphate azabenzotriazole tetramethyl uronium, O-(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-dimethyl formamide, N-methyl-formamide, dimethyl carbonate, diethyl carbonate and a mixture thereof.
11. The process according to claim 1, wherein the tripeptide of formula III is prepared by a process comprising the steps of:
a) activating the carboxylic group of a compound of formula V,

Formula V
wherein R4 is an amine protecting group,
with an activating agent in the presence of a coupling reagent in a solvent to obtain an activated compound of formula VI,

Formula VI
b) condensing the activated compound of formula VI with a dipeptide of formula VII,

Formula VII

in presence of a base in a solvent to obtain a protected tripeptide of formula VIII,

Formula VIII

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

12. The process according to claim 11, wherein R4 is selected from fluorenylmethyloxycarbonyl, tert-butyloxycarbonyl, carboxybenzyl and toluenesulfonyl.
13. The process according to claim 11, wherein
the activating agent in step a) is selected from 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, ethyl 1-hydroxy-1H-1,2,3- triazole-4-carboxylate, and N-hydroxytetrazole;
the coupling reagent in step a) is selected from the group consisting of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, N, N-dicyclohexylcarbodiimide, Oxyma B /diisopropyl carbodiimide, benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate, hexafluorophosphate azabenzotriazole tetramethyl uronium, O-(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, methyl morpholine, 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-dimethyl formamide, N-methyl-formamide, dimethyl carbonate, diethyl carbonate and a mixture thereof.
14. The process according to claim 1, wherein tetrapeptide of formula I is a tetrapeptide of formula Ia,

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

Formula IIa
with a tripeptide of formula IIIa,

Formula IIIa.
16. The process according to any of proceeding claims, wherein the tetrapeptide of formula I contains less than 0.5% of D-isomeric impurity of histidine by HPLC, preferably less than 0.4% of D-isomeric impurity of histidine by HPLC.
17. The process according to any of proceeding claims, wherein the tetrapeptide of formula I has a purity of 98% by HPLC or more, preferably 99% by HPLC or more.
18. A process for the preparation of Liraglutide or pharmaceutically acceptable salts thereof, comprising converting a tetrapeptide of formula I obtained by a process according to any of the preceding claims, to Liraglutide or pharmaceutically acceptable salts thereof.
19. Liraglutide or a pharmaceutically acceptable salt thereof obtained by the process according to claim 18.
20. Liraglutide or a pharmaceutically acceptable salt thereof, according to claim 19 containing less than 0.5% of D-isomeric impurity of histidine by HPLC, preferably less than 0.4% of D-isomeric impurity of histidine by HPLC.

Dated this 13th day of December 2023

Dr. Prachi Tiwari
Sr. Director (Head) – IPM
Fresenius Kabi Oncology Limited