WO2021152622A1 - Procédé amélioré pour la préparation de liraglutide - Google Patents

Procédé amélioré pour la préparation de liraglutide Download PDF

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Publication number
WO2021152622A1
WO2021152622A1 PCT/IN2021/050077 IN2021050077W WO2021152622A1 WO 2021152622 A1 WO2021152622 A1 WO 2021152622A1 IN 2021050077 W IN2021050077 W IN 2021050077W WO 2021152622 A1 WO2021152622 A1 WO 2021152622A1
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WIPO (PCT)
Prior art keywords
resin
liraglutide
fmoc
coupling agent
dmf
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PCT/IN2021/050077
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English (en)
Inventor
Mahender Rao Siripragada
Khalid Anwer Mohammed
Sunil Kumar Gandavadi
Shavali SHAIK
Venkata Ramaiah BOMMENA
Ravi Chandra SAYAVARAPU
Ashok Arige
Bhaskar POOLA
Mohosin Layek
Rehana BEGUM
Mohammed Sharif SHAIK
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Neuland Laboratories Limited
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Publication of WO2021152622A1 publication Critical patent/WO2021152622A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons

Definitions

  • the present invention relates to an improved process for the preparation of Liraglutide having the sequence chemical formula (I).
  • the present invention also relates to novel fragments-2 and -4 which are useful in the preparation of Liraglutide.
  • Fragment -2 Fragment -4:
  • Liraglutide is a long-acting glucagon like peptide agonist developed by Novo Nordisk for the treatment of type-2 diabetes. Liraglutide is marketed under brand name “Victoza” in the form of injection in United States and Europe. Liraglutide is an injectable drug which reduces sugar levels in blood. It is also used to treat obesity.
  • Liraglutide and its process for the preparation is first disclosed in US 6268343. This process leads to the formation of impurities and additional purification techniques required to get pure Liraglutide. This process is highly expensive and commercially not viable.
  • SPPS an amino acid anchored by its C-terminus to an insoluble polymer resin.
  • Protected amino acids are sequentially assembled on resin.
  • the growing chain is bound to the insoluble support, the excess of reagents can be removed by simple filtration.
  • side products can accumulate in addition to side products formed during deprotection.
  • the purification of the final product obtained by SPPS is very difficult and commercially not viable to meet stringent regulatory requirements.
  • hybrid approach both SPPS and LPPS are employed at appropriate places, targeted peptide is assembled by fragment condensation in solution phase whereas the fragments are generated through conventional solid phase peptide synthesis. This approach yields the targeted peptide with good purity and high yields. As the short peptide fragment on solid phase exhibits high coupling efficiency, which makes fragment synthesis scalable to the commercial production. Hybrid approach does not require any advanced equipment for the same.
  • the present invention provides an improved process for the preparation of Liraglutide by a hybrid approach.
  • the present invention provides a cost effective, novel and an efficient process for the preparation of Liraglutide by making appropriate fragments in a solid phase approach followed by condensing these fragments using solution phase approach with higher yields and purity.
  • the present invention relates to an improved process for the preparation of Liraglutide by using three, four or five fragments through hybrid approach. The process will involve the coupling of appropriate fragments in a required sequence, deprotection and condensing them in solution phase, followed by purification on reverse phase HPLC, freeze drying and isolation to get pure liraglutide.
  • the present invention provides a solid phase peptide synthesis for the preparation of Liraglutide compound of formula-I. which comprises: a) synthesis of fragments-1,-2,-3,-4 and -5 on solid support b) anchoring to a resin followed by selective deprotection in presence of a base; c) condensing with a resin obtained in stage -b) in presence of a coupling agent and solvent followed by deprotection in presence of a base; d) condensing with a resin obtained in stage-b) in presence of a coupling agent and solvent followed by deprotection in presence of a base; e) condensing
  • the present invention provides a hybrid approach for the preparation of Liraglutide compound of formula-1.
  • Formula-I which comprises: a) synthesis of fragments- 1, -2, -3, -4 and -5 on solid support b) condensing c) condensing with peptide obtained in stage-b) in presence of coupling agent and solvent followed by deprotection to obtain d) condensing with peptide obtained in stage-c) in presence of a coupling agent followed by deprotection to obtain e) condensing with peptide obtained in step-d) in presence of a coupling agent to obtain protected Liraglutide; f) cleaving the protected Liraglutide using a reagent to obtain crude Liraglutide; g) purifying the crude Liraglutide by preparative HPLC to obtain pure Liraglutide.
  • the present invention relates to novel fragments-2 and -4 which are useful in the preparation of Liraglutide.
  • Fragment -2 Fragment -4:
  • the present invention relates to an improved process for the preparation of novel fragments using solid phase peptide synthesis approach which are useful in the preparation of Liraglutide.
  • the present invention provides a solid phase peptide synthesis for the preparation of which comprises: a) anchoring Fmoc-Val-Ser(Oxa)-OH to a resin in presence of a coupling agent; b) selective deprotection of amino acid using a base; c) coupling of Fmoc-Ser(tBu)-OH to a resin obtained in step-a) in presence of coupling agent in a solvent to obtain dipeptide resin; d) sequential coupling of Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH to the obtained resin in step-a) in presence of a coupling agent; e) cleaving of protected peptide from solid support resin in presence of a reagent to get fragment-2.
  • the present invention provides a solid phase peptide synthesis for the preparation of which comprises: a) anchoring Fmoc-Trp(Boc)-OH to a resin in presence of a coupling agent; b) selective deprotection of amino acid using a base; c) coupling of Fmoc-Ala-OH to a resin obtained in step-a) in presence of coupling agent in a solvent to obtain dipeptide resin; d) sequential coupling of Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc- Lys[Pal(Glu-OtBu)]-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH to the obtained resin in step-a) in presence of a coupling agent; e) cleaving of protected peptide from solid support resin in presence of a reagent
  • the present invention provides a hybrid approach for the preparation of Liraglutide compound of formula-1. which comprises: a) synthesis of fragments-3, -6 and -7 on solid support; b) condensing c) condensing coupling agent to d) cleaving the prote e) purifying the crud
  • EDC.HC1 1 -(dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
  • the present invention provides an improved process for the preparation of Liraglutide by making appropriate fragments on solid support, followed by condensing these fragments using solution phase approach with higher yields and purity.
  • Peptide fragments which are used in the preparation of Liraglutide are as follows.
  • Peptide fragments are prepared by using solid phase peptide synthesis through linear approach.
  • Solid phase peptide synthesis is carried out on an insoluble polymer which is acid sensitive.
  • Acid sensitive resin selected from the group consisting of chloro trityl resin (CTC), sasrin, wang resin, 4-methyltrityl chloride, rink acid resin.
  • CTC resin chloro trityl resin
  • sasrin sasrin
  • wang resin 4-methyltrityl chloride
  • rink acid resin Preferably using CTC resin.
  • the resin used for the synthesis of Liraglutide undergoes swelling in presence of a solvent selected from the group consisting of dichloromethane (DCM), N,N-dimethylformamide (DMF) and N-methyl-2-pyrrolidone or mixture.
  • DCM dichloromethane
  • DMF N,N-dimethylformamide
  • N-methyl-2-pyrrolidone or mixture N-methyl-2-
  • the coupling agent used in the reaction can be selected from the group consisting of Ethylcyano (hydroxyimino)acetate-02)-tri-(l-pyrrolidinyl)-Phosphonium hexa fluorophosphate (PyOxim), ethyl-2 -cyano-2-(hydroxy amino) acetate (Oxyma pure), 0-(benzotriazol-l-yl)-N,N,N’,N'- tetramethyluronium tetrafluoroborate (TBTU), diisopropyl carbodiimide (DIC), 1,3- dicyclohexylcabodiimide (DCC) , 0-(7 -azabenzotriazol- 1 -yl) -N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), l-(dimethyl aminopropyl)-3-ethylcarbodiimide hydrochloride (
  • the base is organic or inorganic base.
  • the inorganic base is selected from the group consisting of potassium carbonate, lithium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, sodium hydroxide, potassium hydroxide, ammonium hydroxide and mixture thereof;
  • the organic base is selected from the group consisting of diisopropyl amine, N,N-diisopropyl ethylamine, triethylamine, dimethylamine, tri methyl amine, isopropyl ethylamine, pyridine, N-methyl morpholine and mixture thereof.
  • Solvents used in this coupling reaction is selected from the group consisting of DMF, DCM, tetrahydrofuran, NMP, DMAC, methanol, ethanol, isopropanol, dichloroethane, 1,4-dioxane, ethyl acetate, acetonitrile, acetone or a mixture thereof.
  • the base used in the deprotection reaction can be selected from group consisting of tert-butyl amine, 20% of 4-methyl piperidine in Dimethyl formamide, 20% of piperidine in Dimethyl formamide and 20% of piperazine in Dimethyl formamide. Preferably using tert-butylamine.
  • the cleavage and global deprotection of the peptide is carried out with a cocktail mixture.
  • the cleavage of peptide from resin involves treating the protected peptide anchored to a resin with an acid having at least a scavenger.
  • the acid used in the cleavage is tri fluoro acetic acid.
  • the scavengers used are selected from the group consisting of TIPS, phenol, thioanisole, water or mixture thereof.
  • Liraglutide is carried out by precipitating with ether solvent.
  • Ether solvent used in this reaction is selected from the group consisting of methyl tert-butyl ether, di ethyl ether, t-butyl methyl ether, diisopropyl ether or mixtures thereof. Finally, lyophilization was carried out to get pure Liraglutide.
  • the present invention provides solid phase peptide synthesis for the preparation of Liraglutide of compound of formula-I.
  • the present invention provides a solid phase peptide synthesis for the preparation of Liraglutide compound of formula-1. which comprises: a) synthesis of fragments- 1, -2, -3, -4 and -5 on solid support b) Anchoring to a resin followed by selective deprotection in presence of a base; c) condensing with a resin obtained in stage -b) in presence of a coupling agent in solvent followed by deprotection in presence of a base; d) condensing with a resin obtained in stage -b) in presence of a coupling agent followed by deprotection in presence of a base; e) condensing in presence of a coupling agent followed by deprotection in presence of a base; f) condensing in presence of a coupling agent to obtain protected Liraglutide; g) cleaving the protected Liraglutide from resin using a reagent to obtain crude Liraglutide; h) purifying by preparative HPLC to obtain
  • step-c) condensation of peptide resin obtained in step-b) with in presence of a coupling agent, preferably using diisopropyl carbodiimide (DIC).
  • a coupling agent preferably using diisopropyl carbodiimide (DIC).
  • the reaction temperature may range from 25 °C to 30°C.
  • the duration of the reaction may range from 1 to 4 hours, preferably for the period of 2-3 hours.
  • the reaction temperature may range from 25 °C to 30°C.
  • step-d) was condensed with peptide resin obtained from step-b) in presence of a coupling agent followed by deprotection using a base.
  • Coupling agent used preferably is DIC and oxyma pure in DMF.
  • Preferable base for deprotection step is 20% of piperidine in Dimethyl formamide.
  • step-e) was condensed with peptide resin obtained from step-b) in presence of a coupling agent followed by deprotection using a base.
  • Coupling agent used preferably is DIC and oxyma pure in DMF.
  • Preferable base for deprotection step is 20% of piperidine in Dimethyl formamide.
  • step-f) condensation of with peptide resin obtained in step-b) in presence of a coupling agent to obtain protected Liraglutide.
  • Coupling agent used preferably is DIC and oxyma pure in DMF
  • step-g) cleavage of protected Liraglutide from solid support resin using a reagent to obtain crude Liraglutide.
  • the preferably used reagent in cleavage step is cocktail mixture of TFA, TIPS, water and DTT.
  • the cleaving of peptide from the resin involves treating the protected peptide anchored to the resin with an acid and at least one scavenger.
  • the peptide cleavage reagent used in the process of the present invention is a cocktail mixture of acid, scavengers and solvents.
  • the reaction temperature may range from 5°C to 30°C, preferably 10-15°C.
  • the duration of the reaction may range from 2 to 6 hours, preferably for the period of 3-4 hours.
  • step-h the obtained crude Liraglutide was purified on reverse phase HPLC using a buffer and a solvent followed by freeze drying to obtain Liraglutide.
  • Buffer used in the reaction is selected from the group consisting of Glacial acetic acid, ammonia solution, Trifluoroacetic anhydride in water, Purified water, Orth phosphoric acid in water, acetonitrile, ethanol, methanol, ethyl acetate, triethylamine in water, ammonium acetate in water, ammonium bicarbonate in water or its mixture.
  • the Fmoc protected amino acids are commercially available or may be prepared according to procedures known in the literature.
  • the coupling reactions may be monitored by Kaiser test.
  • the cleavage of the peptide from the solid support may be accomplished by any conventional methods well known in the art. Accordingly, the present invention provides solution phase peptide synthesis for the preparation of Liraglutide of compound of formula-1.
  • the present invention provides a hybrid approach for the preparation of Liraglutide compound of formula-1. which comprises: a) synthesis of fragments-1, -2, -3, -4 and -5 on solid support b) condensing with in presence of coupling agent and solvent followed by deprotection to obtain c) condensing with peptide obtained in stage -b) in presence of coupling agent followed by deprotection to obtain d) with peptide obtained in stage-c) in presence of a coupling agent followed by deprotection to obtain e) condensing with peptide obtained in step-d) in presence of a coupling agent to obtain protected Liraglutide; f) cleaving the protected Liraglutide using a reagent to obtain crude Liraglutide; g) purifying the crude Liraglutide by preparative HPLC to obtain pure Liraglutide.
  • F Coupling agent used preferably in this step is EDC.HC1 and HOBt in DCM.
  • the reaction temperature may range from 25 °C to 30°C.
  • the duration of the reaction may range from 10 to 30 minutes, preferably for the period of 15-20 minutes.
  • Deprotection of was carried out by using a base.
  • the base used in the reaction is preferably using tert-butyl amine.
  • step-c) was condensed with peptide obtained from step-b) in presence of a coupling agent to obtain
  • Coupling agent used preferably in this step is EDC.HC1 and HOBt in DCM.
  • the reaction temperature may range from 25 °C to 30°C.
  • the duration of the reaction may range from 10 to 30 minutes, preferably for the period of 15-20 minutes.
  • Deprotection of peptide was carried out by using a base.
  • the base used preferably in this reaction is tert-butyl amine.
  • step-d was condensed with peptide obtained from step-c) in presence of a coupling agent to obtain peptide.
  • Coupling agent used preferably in this step is EDC.HC1 and HOBt in DCM.
  • the reaction temperature may range from 25 °C to 30°C.
  • the duration of the reaction may range from 10 to 30 minutes, preferably for the period of 15-20 minutes.
  • step-e Deprotection of obtained peptide was carried out by using a base.
  • the base used preferably in this reaction is tert-butyl amine.
  • step-e was condensed with peptide obtained in step-d) in presence of a coupling agent to obtain protected Liraglutide.
  • Coupling agent used preferably in this step is EDC.HCI and HOBt in DCM.
  • the reaction temperature may range from 25 °C to 30°C.
  • the duration of the reaction may range from 10 to 30 minutes, preferably for the period of 15-20 minutes.
  • step-f protected Liraglutide obtained from step-e) was deprotected using a reagent to obtain crude Liraglutide.
  • the preferably used reagent in cleavage step is cocktail mixture of TLA, TIPS, water and DTT.
  • the deprotection of protected peptide carried out by treating with an acid and at least one scavenger.
  • the peptide cleavage reagent used in the process of the present invention is a cocktail mixture of acid, scavengers and solvents.
  • the reaction temperature may range from 5°C to 30°C, preferably 10-15°C.
  • the duration of the reaction may range from 2 to 6 hours, preferably for the period of 3-4 hours.
  • step-g the obtained crude Liraglutide was purified on reverse phase HPLC using a buffer and a solvent, followed by freeze drying to obtain Liraglutide.
  • Buffer used in this reaction is selected from the group consisting of Glacial acetic acid, ammonia solution, Trifluoroacetic anhydride in water, Purified water, Orth phosphoric acid in water, acetonitrile, Triton-X-100, ethanol, methanol, ethyl acetate, triethyl amine in water, ammonium acetate in water, ammonium bicarbonate in water or its mixture.
  • the Pmoc protected amino acids are commercially available or may be prepared according to procedures known in the literature.
  • the coupling reactions may be monitored by Kaiser test.
  • the cleavage of the peptide from the solid support may be accomplished by any conventional methods well known in the art.
  • the present invention relates to novel fragments-2 and -4 which are useful in the preparation of Liraglutide.
  • the present invention provides a solid phase peptide synthesis for the preparation of which comprises: a) anchoring Fmoc-Val-Ser(Oxa)-OH to a resin in presence of a coupling agent; b) selective deprotection of amino acid using a base; c) coupling of Fmoc-Ser(tBu)-OH to a resin obtained in step-a) in presence of coupling agent in a solvent to obtain dipeptide resin; d) sequential coupling of Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH to the obtained resin in step-a) in presence of a coupling agent; e) cleaving of protected peptide from solid support resin in presence of a reagent to get fragment-2.
  • step-a CTC resin was taken in a SPPS reactor and dichloromethane was added to it.
  • step-b) deprotection was carried out in presence of a base.
  • the base preferably used in this step is 20% piperidine in dime thy lformamide.
  • step-c) condensation of peptide resin obtained in step-a) with Fmoc-Ser(tBu)-OH in presence of a coupling agent.
  • the coupling agent preferably used in the reaction is diisopropyl carbodiimide (DIC).
  • the reaction temperature may range from 25 °C to 30°C.
  • the duration of the reaction may range from 1 to 4 hours, preferably for the period of 2-3 hours.
  • the base preferably used in this reaction is 20% of piperidine in Dimethyl form amide.
  • the reaction temperature may range from 25 °C to 30°C.
  • step-d) Sequential addition of Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH to the obtained resin in step-a) in presence of a coupling agent.
  • Coupling agent preferably used in this step is DIC, oxyma pure in DMF.
  • the base preferable used in deprotection reaction is this step is 20% of piperidine in Dimethyl formamide.
  • step-e cleavage is carried out for protected peptide from solid support resin using a reagent to obtain crude Liraglutide.
  • the preferably used reagent in cleavage step is cocktail mixture of TFA, TIPS, water and DTT.
  • the present invention also provides a solid phase peptide synthesis for the preparation of which comprises: a) anchoring Fmoc-Trp(Boc)-OH to a resin in presence of a coupling agent; b) selective deprotection of amino acid using a base; c) coupling of Fmoc-Ala-OH to a resin obtained in step-a) in presence of coupling agent in a solvent to obtain dipeptide resin; d) sequential coupling of Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc- Lys[Pal(Glu-OtBu)]-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH to the obtained resin in step-a) in presence of a coupling agent; e) cleaving of protected peptide from solid support resin in presence of a rea
  • step-a CTC resin was taken in a SPPS reactor and dichloromethane was added to it. Deprotecting the Fmoc group in presence of a base, preferably using 20% piperidine in dimethylformamide.
  • step-c) condensation of peptide resin obtained in step-b) with Fmoc-Ala-OH in presence of a coupling agent.
  • Coupling agent used preferably is DIC, oxyma pure in DMF.
  • the reaction temperature may range from 25 °C to 30°C.
  • the duration of the reaction may range from 1 to 4 hours, preferably for the period of 2-3 hours.
  • step-d) sequential addition of to the obtained resin in step-a) in presence of a coupling agent.
  • Coupling agent used in preferable in this step is DIC and oxyma pure in DMF.
  • the base used in deprotection step is preferably using 20% of piperidine in Dimethyl formamide.
  • step-e) cleavage is carried out for protected peptide from solid support resin using a reagent to obtain crude Liraglutide.
  • the preferably used reagent in cleavage step is cocktail mixture of TFA, TIPS, water and DTT.
  • the present invention provides a solid phase peptide synthesis for the preparation of
  • step-a CTC resin was taken in a SPPS reactor and dichloromethane was added to it. Fmoc- Arg(Pbf)-OH was added to the resulting reaction mixture in presence of diisopropyl ethylamine.
  • step-b) Deprotecting the Fmoc group in presence of a base, preferably using 20% piperidine in dimethylformamide.
  • the reaction temperature may range from 25°C to 30°C.
  • step-c condensation of peptide resin obtained in step-a) with Fmoc-Gly-OH in presence of coupling agent.
  • the coupling agent preferable used in this step is DIC and oxyma pure in DMF.
  • step-d) Sequential addition of Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc- Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc- Lys[Pal(Glu-OtBu)]-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH to the obtained resin in step-a) in presence of a coupling agent.
  • the coupling agent preferable used in this step is DIC and oxyma pure in DMF
  • the reaction temperature may range from 25 °C to 30°C.
  • the duration of the reaction may range from 1 to 4 hours, preferably for the period of 2-3 hours.
  • the base used in deprotection step is preferably using 20% of piperidine in Dimethyl formamide.
  • step-e partial deprotection is carried out for protected peptide from solid support resin using a reagent to obtain Fragment-6.
  • Reagent used in partial deprotection is selected from the group consisting of TFA, TIPS, Water, DTT, Thioanisole, EDT, DMS, cresol, phenol, thiocresol, ammonium iodide, 2,2'-(ethylene dioxy)diethane or its mixture.
  • TFA in dichloromethane.
  • step-f coupling of H-Gly-OtBu.HCl to the 14 amino acid peptide chain obtained in step-e) in presence of coupling agent.
  • Coupling agent preferably used in this step is EDC.HC1, HOAt in DMF.
  • reagent used for deprotection of peptide obtained in step-f) is preferably using palladium on carbon.
  • step-h) coupling of Pal-Glu(OSu)-OtBu to 15 amino acid peptide chain in step-g) in presence of a base like sodium carbonate solution or potassium carbonate solution to obtain protected 15 amino acid peptide chain
  • step-i) deprotection of protected 15 amino acid peptide chain in step-h) is carried out in presence of tert-butyl amine.
  • the present invention provides a solid phase peptide synthesis for the preparation of Boc-His(Trt)-Ala-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)- Val-Ser(Oxa)-OH (Fragment- 7)
  • step-a CTC resin was taken in a SPPS reactor and dichloromethane was added to it. Fmoc-Val- Ser(Oxa)-OH was added to the resulting reaction mixture in presence of coupling agent and diisopropyl ethylamine.
  • the coupling agent preferably used in this step is DIC and oxyma pure in DMF.
  • step-b Deprotecting the Fmoc group in presence of a base.
  • the base used in this step is preferably using 20% piperidine in dimethylformamide.
  • the reaction temperature may range from 25 °C to 30°C.
  • the duration of the reaction may range from 1 to 4 hours, preferably for the period of 2-3 hours.
  • step-c) condensation of peptide resin obtained in step-b) with Fmoc-Ser(tBu)-OH in presence of coupling agent.
  • the coupling agent preferably used in this step is DIC and oxyma pure in DMF.
  • step-d) Sequential addition of Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc- Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH, Boc-His(Trt)-OH to the obtained resin in step-a) in presence of a coupling agent.
  • the coupling agent preferably used in this step is DIC and oxyma pure in DMF.
  • the reaction temperature may range from 25 °C to 30°C.
  • the duration of the reaction may range from 1 to 4 hours, preferably for the period of 2-3 hours.
  • Deprotection of obtained peptide was carried out using base, preferably using 20% of piperidine in Dimethyl formamide.
  • step-e cleavage is carried out for protected peptide from solid support resin using a reagent to obtain Fragment -7.
  • the preferably used reagent in cleavage step is TFA in dichloromethane.
  • the present invention provides solution phase peptide synthesis for the preparation of Liraglutide of compound of formula-I by using three fragment approach.
  • the present invention provides a hybrid approach for the preparation of Liraglutide compound of formula-1. which comprises: a) synthesis of fragments-3, -6 and -7 on solid support; b) condensing c) condensing with peptide obtained in step-b) in presence of a coupling agent to obtain protected Liraglutide; d) cleaving the protected Liraglutide using a reagent to obtain crude Liraglutide; e) purifying the crude Liraglutide by preparative HPLC to obtain pure Liraglutide.
  • step-a) fragments-3, -6 and -7 are prepared by solid phase peptide synthesis. in presence of coupling agent obtain protected in in-situ manner. Further, it is deprotected in presence of a base to obtain
  • the coupling agent preferably used in this reaction is EDC.HC1 and HOAt in DCM.
  • the reaction temperature may range from 25 °C to 30°C.
  • the duration of the reaction may range from 10 to 30 minutes, preferably for the period of 15-20 minutes.
  • the base preferably used is tert-butylamine.
  • step-c) was condensed with peptide obtained from step-b) in presence of a coupling agent to obtain protected Liraglutide.
  • Coupling agent preferably used in this reaction is EDC.HC1 and HO At in DCM.
  • the reaction temperature may range from 25 °C to 30°C.
  • the duration of the reaction may range from 1 to 3 hours, preferably for the period of 1-2 hours.
  • step-d) protected Liraglutide obtained from step-c) was deprotected using a reagent to obtain crude Liraglutide.
  • the preferably used reagent in cleavage step is cocktail mixture of TLA, TIPS, water and DTT.
  • the deprotection of protected peptide carried out by treating with an acid and at least one scavenger.
  • the peptide cleavage reagent used in the process of the present invention is a cocktail mixture of acid, scavengers and solvents.
  • the reaction temperature may range from 5°C to 30°C, preferably 10-15°C.
  • the duration of the reaction may range from 2 to 6 hours, preferably for the period of 3-4 hours.
  • step-e the obtained crude Liraglutide was purified on reverse phase HPLC using a buffer and a solvent, followed by freeze drying to obtain Liraglutide.
  • the buffer used in the reaction is selected from the group consisting of Glacial acetic acid, ammonia solution, Trifluoroacetic anhydride in water, Purified water, Orth phosphoric acid in water, acetonitrile, Triton-X-100, ethanol, methanol, ethyl acetate, triethyl amine in water, ammonium acetate in water, ammonium bicarbonate in water or its mixture.
  • the Pmoc protected amino acids are commercially available or may be prepared according to procedures known in the prior art literature.
  • the coupling reactions may be monitored by Kaiser test.
  • the cleavage of the peptide from the solid support may be accomplished by any conventional methods well known in the art.
  • the present invention provides a hybrid approach for the preparation of Liraglutide compound of formula-I. which comprises: a) Synthesis of fragments- 1, -2, -3 and -6 on solid support; b) condensing with in presence of coupling agent and solvent in in-situ manner followed by deprotection in presence of base to obtain c) condensing with peptide obtained in step-b) in presence of a coupling agent in in-situ manner followed by deprotection in presence of base to obtain d) condensing with peptide obtained in step-c) in presence of a coupling agent in in-situ manner followed by deprotection in presence of base to obtain protected Liraglutide; e) cleaving the protected Liraglutide using a reagent to obtain crude Liraglutide; f) purifying the crude Liraglutide by preparative HPLC to obtain pure Liraglutide.
  • the coupling agent preferably used in this step is EDC.HC1 and HOAt in DCM.
  • the reaction temperature may range from 25 °C to 30°C.
  • the duration of the reaction may range from 1 to 3 hours, preferably for the period of 1-2 hours.
  • Deprotection of was carried out by using a base in in-situ manner.
  • the base used in the reaction is preferably using tert- butylamine.
  • step-c was condensed with peptide obtained from step-b) in presence of a coupling agent to obtain Fmoc protected
  • the coupling agent preferably used in this step is EDC.HC1 and HOAt in DCM.
  • the reaction temperature may range from 25 °C to 30°C.
  • the duration of the reaction may range from 1 to 3 hours, preferably for the period of 1-2 hours.
  • Deprotection of was carried out by using a base in in- situ manner, preferably base used in deprotection reaction is tert-butylamine.
  • step-d was condensed with the peptide obtained in step- c) in presence of a coupling agent and solvent to obtain protected Eiraglutide.
  • the coupling agent preferably used in this step is EDC.HC1 and HOAt in DCM.
  • the reaction temperature may range from 25 °C to 30°C.
  • the duration of the reaction may range from 1 to 3 hours, preferably for the period of 1-2 hours.
  • protected Liraglutide obtained from step-d) was deprotected using a reagent to obtain crude Liraglutide.
  • the deprotection of protected peptide carried out by treating with an acid and at least one scavenger.
  • the peptide cleavage reagent used in the process of the present invention is a cocktail mixture of acid, scavengers and solvents.
  • the preferably used reagent in cleavage step is cocktail mixture of TFA, TIPS, water and DTT.
  • the reaction temperature may range from 5°C to 30°C, preferably 10-15°C.
  • the duration of the reaction may range from 2 to 6 hours, preferably for the period of 3-4 hours.
  • step-f the obtained crude Liraglutide was purified on reverse phase F1PLC using a buffer and a solvent, followed by freeze drying to obtain Liraglutide.
  • Example- 1 Process for the preparation of Liraglutide by using soild phase peptide synthesis approach
  • CTC resin 50 grams was taken in a SPPS reactor and dichloromethane was added and allowed it to swell for 10 minutes.
  • Fmoc-Glu(OtBu)-OH (76.5 grams) was dissolved in DMF and stirred for 10 minutes. DIC (22.72 grams) and oxyma (25.56 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with DMF, isopropanol and dichlorome thane. The resulting resin was deblocked with 20% piperidine in DMF for 10 minutes and washed with DMF.
  • Fmoc-Ala-OH (56.03 grams) was dissolved in DMF and stirred for 10 minutes. DIC (22.72 grams) and oxyma (25.56 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was drained and washed with DMF, isopropanol and dichlorome thane. The resulting resin was deblocked with 20% piperidine in DMF.
  • Boc-Fhs(Trt)-OH (89.46 grams) was dissolved in DMF and stirred for 10 minutes. DIC (22.72 grams) and oxyma (25.56 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was washed with DMF, isopropanol and dichloromethane.
  • Stage-2 Synthesis of Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-OH [Fragment-2]
  • Step-A CTC resin (50 grams) was taken in a SPPS reactor and dichloromethane was added and allowed it to swell for 10 minutes.
  • the above resin was deblocked with 20% piperidine in DMF for 10-15 minutes and washed with DMF.
  • Fmoc-Ser(tBu)-OH (18.49 grams) was dissolved in DMF and stirred for 10 minutes. DIC (6 grams) and oxyma (6.82 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with DMF, isopropanol and dichloromethane. The resulting resin was deblocked with 20% piperidine in DMF for 10 minutes and washed with DMF.
  • CTC resin 50 grams was taken in a SPPS reactor and dichloromethane was added and allowed it to swell for 10 minutes.
  • the above resin was deblocked with 20% piperidine in DMF for 10-15 minutes and washed with DMF.
  • Fmoc-Tyr(tBu)-OH (69 grams) was dissolved in DMF and stirred for 10 minutes. DIC (18.92 grams) and oxyma (21.3 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was washed with DMF, isopropanol and dichloromethane.
  • Fmoc-Ser(tBu)-OH (57.51 grams) was dissolved in DMF and stirred for 10 minutes. DIC (18.92 grams) and oxyma (21.3 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was washed with DMF, isopropanol and dichloromethane.
  • the above resin was deblocked with 20% piperidine in DMF for 10-15 minutes and washed with DMF.
  • Fmoc-Ile-OH (10.60 grams) was dissolved in DMF and stirred for 10 minutes. DIC (3.79 grams) and oxyma (4.26 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was drained and washed with DMF, isopropanol and dichloromethane. The resulting resin was deblocked with 20% piperidine in DMF.
  • Fmoc-Glu(OtBu)-OH (12.76 grams) was dissolved in DMF and stirred for 10 minutes. DIC (3.79 grams) and oxyma (4.26 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction, the resin was washed with DMF, isopropanol and dichloromethane.
  • Fmoc-Ala-OH (9.33 grams) was dissolved in DMF and stirred for 10 minutes. DIC (3.79 grams) and oxyma (4.26 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction, the resin was washed with DMF, isopropanol and dichloromethane.
  • Fmoc-Ala-OH (9.33 grams) was dissolved in DMF and stirred for 10 minutes. DIC (3.79 grams) and oxyma (4.26 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction, the resin was washed with DMF, isopropanol and dichloromethane.
  • CTC resin 50 grams was taken in a SPPS reactor and dichloromethane was added and allowed it to swell for 10 minutes.
  • the above resin was deblocked with 20% piperidine in DMF for 10-15 minutes and washed with DMF.
  • Resin and peptide obtained from step-G were taken in a SPPS reactor and N,N-Dimethyl formamide was added and allowed it to swell for 10 minutes.
  • Gly-OtBu. F1C1 (6.57 grams) is added in presence of EDC.F1C1 (7.59 grams) and NMM (3.48 grams) at 25-30°C and stirred for 2-3 hours at the same temperature. Cooled the resulting reaction mixture and water was added to it. Filtered the precipitated solid and washed with water.
  • CTC resin was taken in a SPPS reactor and dichlorome thane was added and allowed it to swell for 10 minutes. and Diisopropyl ethylamine in dry dichloromethane were added to the resin and stirred for 2 hours at 25-30°C. The progress of coupling was monitored by Kaiser test. After completion of reaction, the resulting resin was deblocked with 20% piperidine in DMF.
  • Step-B (Fragment-4) was dissolved in DMF and stirred for 10 minutes. DIC and oxyma were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction, the resin was washed with DMF. The resulting resin was deblocked with 20% piperidine in DMF followed by washing with DMF.
  • Step-C was dissolved in DMF and stirred for 10 minutes. DIC and oxyma were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction, the resin was washed with DMF. The resulting resin was deblocked with 20% piperidine in DMF followed by washing with DMF.
  • Step-D was dissolved in DMF and stirred for 10 minutes. DIC and oxyma were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction, the resin was washed with DMF. The resulting resin was deblocked with 20% piperidine in DMF followed by washing with DMF.
  • Step-E was dissolved in DMF and stirred for 10 minutes. DIC and oxyma were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction, the resin was washed with DMF.
  • Step-F Crude Liraglutide obtained in step-F was dissolved in 0.5 M ammonium formate and loaded on to preparative C8 column (50 X 250 mm, 100 A°).
  • the peptide was purified using a linear gradient of aqueous TFA (0.1%) and acetonitrile: methanol (8:1, 0.1% TFA) from 40% to 90% over 60 minutes.
  • the pure fraction containing the Liraglutide was pooled. Volatiles were removed under reduced pressure and aqueous layer was lyophilized to give Liraglutide as a powder.
  • the resulting peptide was analysed by RP-HPLC and confirmed by MALDI or LC-MS.
  • Example-2 Process for the preparation of Liraglutide by using solution phase peptide synthesis approach
  • Step-A (Fragment-4) obtained from stage -4 of example- 1 was dissolved in DMF and stirred for 10 minutes at 25-30°C.
  • Step-B (Fragment-3) obtained from stage-3 of example-1 was dissolved in DMF and stirred for 10 minutes.
  • Fl-Protected 16 amino acid peptide obtained in step-A was added in presence of EDC.F1C1 and FIOBT in DCM at 25-30°C and stirred for 15-20 minutes at the same temperature.
  • Precipitated solid was filtered and washed with water and hexane followed by dried under vacuum for 2 hours.
  • the resulting protected peptide was deprotected with tert -butyl amine and n-heptane in DMF. Filtered the precipitated solid and washed with water, hexane and methanol to get
  • Step-C (Fragment -2) obtained from stage-2 of example-1 was dissolved in DMF and stirred for 10 minutes. Peptide obtained in step-B was added in presence of EDC.F1C1 and FIOBT in DCM at 25-30°C and stirred for 15-20 minutes at the same temperature. Precipitated solid was filtered and washed with water and hexane followed by dried under vacuum for 2 hours. The resulting protected peptide was deprotected with tert-butyl amine and n-heptane in DMF. Filtered the precipitated solid and washed with water, hexane and methanol to get
  • Step-D (Fragment-1) obtained from stage-1 of example-1 was dissolved in DMF and stirred for 10 minutes. Peptide obtained in step-C was added in presence of EDC.F1C1 and FIOBT in DCM at 25-30°C and stirred for 15-20 minutes at the same temperature. Precipitated solid was filtered and washed with water and hexane followed by dried under vacuum for 2 hours to get Boc -protected peptide.
  • Stage-1 solid phase peptide synthesis of
  • Fmoc-Ser(tBu)-OH (18.49 grams) was dissolved in DMF and stirred for 10 minutes. DIC (6 grams) and oxyma (6.82 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with DMF, isopropanol and dichloromethane. The resulting resin was deblocked with 20% piperidine in DMF for 10 minutes and washed with DMF.
  • Fmoc-Phe-OH (18.60 grams) was dissolved in DMF and stirred for 10 minutes. DIC (6.0 grams) and oxyma (6.82 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was washed with DMF, isopropanol and dichlorome thane. The resulting resin was deblocked with 20% piperidine in DMF.
  • Fmoc-Gly-OFi (47.57 grams) was dissolved in DMF and stirred for 10 minutes. DIC (6.0 grams) and oxyma (6.82 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was washed with DMF, isopropanol and dichloromethane. The resulting resin was deblocked with 20% piperidine in DMF.
  • Fmoc-Glu(OtBu)-OFi 76.5 grams was dissolved in DMF and stirred for 10 minutes.
  • DIC 22.72 grams
  • oxyma 25.56 grams
  • the progress of coupling was monitored by Kaiser tests.
  • the resin was drained and washed with DMF, isopropanol and dichloromethane. The resulting resin was deblocked with 20% piperidine in DMF for 10 minutes and washed with DMF.
  • Fmoc-AIa-OFi (56.03 grams) was dissolved in DMF and stirred for 10 minutes. DIC (22.72 grams) and oxyma (25.56 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was drained and washed with DMF, isopropanol and dichloromethane. The resulting resin was deblocked with 20% piperidine in DMF.
  • Boc-His(Trt)-OH (89.46 grams) was dissolved in DMF and stirred for 10 minutes. DIC (22.72 grams) and oxyma (25.56 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was washed with DMF, isopropanol and dichloromethane.
  • CTC resin 50 grams was taken in a SPPS reactor and dichloromethane was added and allowed it to swell for 10 minutes.
  • the above resin was deblocked with 20% piperidine in DMF for 10-15 minutes and washed with DMF.
  • Fmoc-Gly-OFi (44.59 grams) was dissolved in DMF and stirred for 10 minutes. DIC (23.2 grams) and oxyma (21.3 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with DMF, isopropanol and dichloromethane. The resulting resin was deblocked with 20% piperidine in DMF for 10 minutes and washed with DMF.
  • Fmoc-Ala-OH (9.33 grams) was dissolved in DMF and stirred for 10 minutes.
  • DIC (3.79 grams) and oxyma (4.26 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step -A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with DMF, isopropanol and dichloromethane. The resulting resin was deblocked with 20% piperidine in DMF for 10 minutes and washed with DMF.
  • Fmoc-Ile-OH (10.60 grams) was dissolved in DMF and stirred for 10 minutes. DIC (3.79 grams) and oxyma (4.26 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was drained and washed with DMF, isopropanol and dichloromethane. The resulting resin was deblocked with 20% piperidine in DMF.
  • Fmoc-Ala-OH (9.33 grams) was dissolved in DMF and stirred for 10 minutes. DIC (3.79 grams) and oxyma (4.26 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction, the resin was washed with DMF, isopropanol and dichloromethane. The resulting resin was deblocked with 20% piperidine in DMF.
  • Fmoc-Ala-OH (9.33 grams) was dissolved in DMF and stirred for 10 minutes. DIC (3.79 grams) and oxyma (4.26 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction, the resin was washed with DMF, isopropanol and dichloromethane. The resulting resin was deblocked with 20% piperidine in DMF.
  • H-Gly-OtBu.HCl was dissolved in DMF and stirred for 10 minutes. EDC.HC1 and HOAt were added to the resulting reaction mixture and stirred for 1 -2 hours at the same temperature. It was added to the resin obtained in step-A and stirred for 1-2 hours. Protected 15 amino acid peptide chain was deprotected with 5% palladium on carbon to obtain 15 amino acid peptide chain.
  • Pal-Glu(OSu)-OtBu was dissolved in DMF and added to 15 amino acid peptide chain in presence of sodium carbonate solution and stirred for 4-5 hours at room temperature.
  • the resulting peptide is precipitated with water, hexane and dried under vacuum at 45° C for 2-3 h to obtain protected 15 Amino acid peptide chain.
  • the obtained peptide was dissolved in DMF and cooled to 5-10°C. Tert- butyl amine was added to the resulting solution. Water was added to the resulting reaction mixture to obtain Fragment-6.
  • Step-A (Fragment-6) was dissolved in DMF and stirred for 10 minutes at 25-30°C. (1.37 grams) [Fragment-3 (obtained from stage-3 of example-1, EDC.HC1 and HOAT in DCM were added to the resulting reaction mixture at 25-30°C and stirred for 15-20 minutes at the same temperature. Precipitated solid was filtered and washed with water and hexane. The resulting protected peptide was deprotected with tert-butylamine. Filtered the precipitated solid and washed with water and hexane to get
  • Step-B and stirred for 3-6 hours at the same temperature. Chilled DIPE was added to the resulting mixture and stirred for 2 hours. The precipitated solid was filtered and washed with DCM followed by DIPE to get crude Liraglutide.
  • Example-4 Process for the preparation of Liraglutide by using hybrid approach [four fragment approach]
  • Step-A (Lragment-6) obtained from stage-2 of example-3 was dissolved in DML and stirred for 10 minutes at 25-30°C. (Lragment-3) obtained from stage-3 of example- 1, EDC.HC1 and HOAT in DCM were added to the resulting reaction mixture at 25-30°C and stirred for 15-20 minutes at the same temperature. Precipitated solid was filtered and washed with water and hexane. The resulting protected peptide was deprotected with tert-butylamine. Liltered the precipitated solid and washed with water and hexane to get Step-B: (Fragment-2) was dissolved in DMF and stirred for 10 minutes.
  • H-Protected 20 amino acid peptide obtained in step-A was added in presence of EDC.F1C1 and FIOAT in DCM at 25-30°C and stirred for 15-20 minutes at the same temperature. Precipitated solid was filtered and washed with water and hexane. The resulting protected peptide was deprotected with tert-butylamine. Filtered the precipitated solid and washed with water and hexane to get
  • Step-C (Fragment-1) was dissolved in DMF and stirred for 10 minutes. H -Protected amino acid peptide obtained in step-B was added in presence of EDC.F1C1 and FIOAT in DCM at 25-30°C and stirred for 15-20 minutes at the same temperature. Precipitated solid was filtered and washed with water and hexane followed by dried under vacuum for 2 hours. The resulting protected peptide was cleaved with a cocktail mixture of TFA, TIPS, water and DTT in presence of DCM at 10-15°C and stirred for 3-6 hours at the same temperature. Chilled DIPE was added to the resulting mixture and stirred for 2 hours. The precipitated solid was filtered and washed with DCM followed by DIPE to get crude Liraglutide.

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Abstract

La présente invention concerne un procédé amélioré pour la préparation de liraglutide ayant la formule développée (I). His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Glu-Palmitoyl)-Glu-Phe-lle-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH. Formule-I. La présente invention concerne de nouveaux fragments 2 et 4 qui sont utiles dans la préparation de liraglutide. Fragment 2 : Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-OH Fragment 4 : Fmoc-Gln(Trt)-Ala-Ala-Lys(palmityl-γ-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-OH La présente invention concerne également les fragments suivants qui sont utiles dans la préparation de liraglutide. Fragment 1 : Boc-His(Trt)-Ala-Glu(OtBu)-Gly-OH Fragment 2 : Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-OH Fragment 3 : Fmoc-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH Fragment 4 : Fmoc-Gln(Trt)-Ala-Ala-Lys(palmityl-γ-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-OH Fragment 5 : Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-OtBu Fragment 6 : H-Gln(Trt)-Ala-Ala-Lys(palmityl-γ-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OtBu Fragment 7 : Boc-His(Trt)-Ala-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-OH
PCT/IN2021/050077 2020-01-27 2021-01-25 Procédé amélioré pour la préparation de liraglutide WO2021152622A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023234860A1 (fr) * 2022-06-01 2023-12-07 Scinopharm Taiwan Ltd. Procédé de préparation du glucagon-like peptide-1

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016046753A1 (fr) * 2014-09-23 2016-03-31 Novetide, Ltd. Synthèse de peptides glp-1
WO2016067271A1 (fr) * 2014-10-31 2016-05-06 Auro Peptides Ltd Procédé de préparation de liraglutide

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016046753A1 (fr) * 2014-09-23 2016-03-31 Novetide, Ltd. Synthèse de peptides glp-1
WO2016067271A1 (fr) * 2014-10-31 2016-05-06 Auro Peptides Ltd Procédé de préparation de liraglutide

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023234860A1 (fr) * 2022-06-01 2023-12-07 Scinopharm Taiwan Ltd. Procédé de préparation du glucagon-like peptide-1

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