WO2023105497A1 - Synthèse d'analogues de glp-1 - Google Patents

Synthèse d'analogues de glp-1 Download PDF

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Publication number
WO2023105497A1
WO2023105497A1 PCT/IB2022/062039 IB2022062039W WO2023105497A1 WO 2023105497 A1 WO2023105497 A1 WO 2023105497A1 IB 2022062039 W IB2022062039 W IB 2022062039W WO 2023105497 A1 WO2023105497 A1 WO 2023105497A1
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Prior art keywords
amino acid
acid sequence
peptide fragment
sequence seq
peptide
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PCT/IB2022/062039
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English (en)
Inventor
Atul Kasturchand Godha
Sathya Vanganur NARAYANASWAMY
Areti Venkata Bhagyaraj
Aslam Jahangir Bavadekar
Rudresh LAKSHMANAPPA
Sheiksyedali NAGOOR
Ranjithkumar MURUGAN
Amol Vasantrao Gadakh
Ganesh Sambasivam
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Anthem Biosciences Pvt. Ltd.
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Publication of WO2023105497A1 publication Critical patent/WO2023105497A1/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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0808—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu

Definitions

  • the present invention relates generally to field of synthetic organic chemistry and preparation of peptides of pharmaceutical significance. More particularly, the disclosure provides synthesis method of glucagon like peptides via fragment approach and sequential amino acids couplings.
  • Glucagon-like peptide- 1 is a naturally occurring 30 or 31 amino acid long peptide hormone deriving from the tissue-specific posttranslational processing of the proglucagon peptide, which stimulates insulin release and decreases the level of the anti-insulin hormone glucagon in response to increases in blood sugar levels.
  • Glucagon-like peptide- 1 is typically produced by yeast through recombinant gene technology.
  • GLP- 1 analogues include Exenatide, Liraglutide, Lixisenatide, Albiglutide, Dulaglutide, Semaglutide and Taspoglutide.
  • the synthesis strategies adopted in the prior art for the glucagon-like peptide- 1 peptide includes different approaches such as uses of different/multiple resins, coupling reagents, fragments, different protecting group for amino acids.
  • Liraglutide is an analogue of glucagon-like peptide- 1 containing a backbone of 31 amino acids, wherein the Lys-20 is condensed with Glu-Pal group. Liraglutide is produced by covalently linking glucagon-like peptide- 1 to a fatty acid. It is a once- daily injectable and has the effects of lowering blood sugar level, reducing body weight, promoting islet cell regeneration, as well as protecting cardiovascular system.
  • Semaglutide is a next generation glucagon-like peptide- 1 analogue as a once- weekly injection. Semaglutide shares a similar backbone to Liraglutide, with Ala2 being substituted by Aib, and Lys20 is side modification moiety derivatized with N-(17-carboxy-loxoheptadecyl)-L-y-glutamyl-2-[2-(2-aminoethoxy] ethoxy] acetyl-2-[2-(2-aminoethoxy) ethoxy] acetyl.
  • CN106478806A provides uses of Dde-Lys(Fmoc)-OH for branching after sequential synthesis of peptide backbone. After deprotection of -Fmoc protecting group of Lysine branching is carried out, Dde of lysine is de -protected with hydrazine hydrate in DMF and further amino acids are coupled.
  • CN105753964A describes uses of different resins for linear synthesis such as Cl- trityl resin,
  • HMPB-AM resin Wang Resin in combination with Dde-Lys(Fmoc)-OH, Fmoc- Lys(Alloc)OH as branching points on the linear backbone.
  • methyl ester of linker is synthesized using resin.
  • dipeptide Ala-Ala and Ser-Ser fragments are used to avoid defective peptide formation.
  • Methyl ester of linker is hydrolysed by LiOH to afford pure peptide.
  • CN103848910A describes sequential synthesis of backbone peptide which includes Fmoc-Lys(Mmt)-OH and Boc-His-OH for branching.
  • WO 2019/069274 Al provides uses of different fragments (1-19) with a fragment (20-31), or a fragment (1-18) with a fragment (19-31). Another preferred coupling of fragment (1-17) with a fragment (18-31). In addition, with carboxyl terminal amino acid of the first fragment is a Leu residue coupling a fragment (1-14) with a fragment (15-31).
  • Liraglutide and Semaglutide in the above referred patent applications, are known to contain positions in their sequences where the coupling and deprotection steps become difficult. This results in the formation of several undesirable by products which lower the yield and make purification more difficult.
  • One particular difficulty with the synthesis lies in the incorporation of Lys (20) into the growing peptide chain of Liraglutide and Semaglutide due to presence of its side chain part, which often results in racemization and the formation of unwanted peptide.
  • the above prior arts disclose opting out synthesis of main peptide backbone in the initial step followed by incorporation of side chain at Lys (20) position in the second step to form the desired GLP-1 peptide.
  • CN111732650A discloses a method for preparing Semaglutide by combining a continuous flow solid-phase synthesis system.
  • the method comprises the following steps of firstly, introducing Octadecanedioic(OtBu) acid-gamma-Glu-(OtBu)- AEEA-AEEA into a side chain of Lys20, and then completing a strategy of a main chain, wherein Fmoc-Gly-resin is adopted as a main chain structure, and continuous flow solid-phase synthesis is adopted for polypeptide fragment and Semaglutide peptide chain growth.
  • the method further involves purification of peptide by mixing the crude peptide in a solution of acetonitrile and water, carrying out purification by using a C18 or C8 preparation column to remove main impurities before and after the product, followed by fine purification to remove micro impurities, and carrying out salt conversion and freeze-drying to obtain the product with the purity of 99.62%.
  • CN111732650A discloses obtaining pure Semaglutide, it uses pseudo serine in the synthesis of one of the peptide fragments, which is not only expensive from industrial point of view, also not amenable from scale up point of view.
  • US20190177392A1 discloses methods for synthesis of GLP-1 peptides, such as Liraglutides and Semaglutides, and a method for purifying Liraglutide where crude Liraglutide has 56.5% purity, which after several HPLC purification cycles reaches to the 98.5% purity level.
  • W02020190757A1 discloses methods for the preparation and purification of Semaglutide and intermediates, where the crude Semaglutide obtained have 60.1% purity.
  • the purification of Semaglutide was done by 3 purification systems on preparative HPLC column, which resulted in 98.9% purity.
  • RP HPLC was used to obtain 99.7% purity.
  • US20200317721A1 discloses a method for preparing GLP-1 and GLP-2 peptides, which comprises following multiple steps of purification involving HPLC for obtaining more than 99% purity in peptides.
  • US20210009631A1 discloses methods and compounds for the solid phase synthesis of Semaglutide which results in 42.1% pure crude Semaglutide which after more than one step of dimensional purification by RP-HPLC.
  • WO2022018748A1 discloses improved and effective purification methods for Semaglutide by following RP-HPLC purification at three stages under varying conditions. First purification is followed in basic condition, followed by second in acidic and then third again in basic condition. After multiple RP-HPLC approaches Semaglutide obtained had more than 98% purity.
  • It is an object of the present invention is to provide an improved and industrially advantageous method for preparation of peptides of pharmaceutical and commercial significance via fragment approach.
  • Figure 1 illustrates synthesis of peptide fragment of SEQ ID NO: 1 (Fragment IG)
  • Figure 2 illustrates synthesis of peptide fragment of SEQ ID NO: 2 (Fragment SG)
  • FIG. 3 illustrates synthesis of peptide fragment Fragment BG
  • Figure 4 illustrates synthesis of peptide fragment of SEQ ID NO: 3 (Fragment YF)
  • Figure 5 illustrates synthesis of peptide fragment of SEQ ID NO: 4 (Fragment YA)
  • Figure 6 illustrates synthesis of Semaglutide involving coupling of peptide fragments of SEQ ID NO: 1, i.e., P-Ile-Ala-Trp (Boc)-Leu-Val-Arg (Pbf)-Gly-OH; SEQ ID NO: 2, i.e., P-Ser (tBu) - Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH and Fragment BG P-Aib-Glu (OtBu)-Gly-OH.
  • Figure 7 illustrates synthesis of Semaglutide involving coupling of peptide fragments of SEQ ID NO: 1, i.e., P-Ile-Ala-Trp (Boc)-Leu-Val-Arg (Pbf)-Gly-OH; SEQ ID NO: 3, i.e., P-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala- Lys(AEEAc-AEEAc -y-Glu-17-Carboxyheptadecanoyl)-Glu(OtBu)-Phe-OH; and Fragment BG, i.e., P-Aib-Glu (OtBu)-Gly-OH.
  • SEQ ID NO: 1 i.e., P-Ile-Ala-Trp (Boc)-Leu-Val-Arg (Pbf)-Gly-OH
  • SEQ ID NO: 3 i.e.,
  • Figure 8 illustrates synthesis of Semaglutide involving coupling of peptide fragments of SEQ ID NO: 1, i.e., P-Ile-Ala-Trp (Boc)-Leu-Val-Arg (Pbf)-Gly-OH; SEQ ID NO: 4, i.e., P-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-OH and Fragment BG, i.e., P-Aib-Glu (OtBu)-Gly-OH.
  • a method of preparing a GLP-1 peptide comprising the steps of: a. providing a first peptide fragment having amino acid sequence namely P -Arg (Pbf)-Gly-resin; b. deprotecting said first peptide fragment in presence of deprotecting agent and coupling deprotected first peptide fragment with peptide fragment having amino acid sequence SEQ ID NO:1 to form peptide fragment having amino acid sequence SEQ ID NOG in presence of a coupling agent, a base and a solvent, c.
  • a method of preparing GLP- 1 peptide comprising the steps of: i. providing a first peptide fragment having amino acid sequence namely P-Arg (Pbf)-Gly-resin, ii. deprotecting said first peptide fragment in presence of deprotecting agent and coupling deprotected first peptide fragment with peptide fragment having amino acid sequence SEQ ID NO:1 to form peptide fragment having amino acid sequence SEQ ID NO:5 in presence of a coupling agent, a base and a solvent, iii.
  • a method of preparing a GLP-1 peptide comprising the steps of: a. . providing a first peptide fragment having amino acid sequence namely P - Arg (Pbf)-Gly-resin, b. . deprotecting said first peptide fragment in presence of deprotecting agent and coupling deprotected first peptide fragment with peptide fragment having amino acid sequence SEQ ID NO: 1 to form peptide fragment having amino acid sequence SEQ ID NO:5 in presence of a coupling agent, a base and a solvent, c.
  • Described herein are the novel peptide fragments having amino acid sequences and methods for synthesizing GLP-1 peptide by using those peptides.
  • peptide fragments having amino acid sequences SEQ ID NO: 1 (Fragment IG), SEQ ID NO:2 (Fragment SG), Fragment BG, SEQ ID NO: 3 (Fragment YF) and SEQ ID NO: 4 (Fragment YA) for the synthesis of GLP-1 peptides.
  • the present invention provides
  • SEQ ID NO: 4 (Fragment YA): P-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala- OH wherein P is N terminal protecting group.
  • the amino acids and the peptide fragments are provided with a protecting group.
  • the protecting groups for preventing reaction with N terminal group of amino acids are selected from Fmoc, Boc, Cbz, Bpoc.
  • the present invention provides method of preparation of GEP-1 peptide involving one or more steps of coupling of peptide fragments with amino acids.
  • the present invention provides a method of preparation of GEP-1 peptide which comprises at least one of said peptide fragment sequences selected from SEQ ID NO: 1 (Fragment IG), SEQ ID NO: 2 (Fragment SG), (Fragment BG), SEQ ID NOG (Fragment YF), and SEQ ID NO: (Fragment YA) or combinations thereof couple with one or more amino acids to form GLP-1 peptide.
  • the GLP-1 peptides formed by the coupling of one or more peptide fragments sequences selected from the above group with amino acids are found to follow less complicated and economical purification procedures, which do not involve multiple steps of HPLC.
  • the GLP-1 peptides formed by the above method are selected from Exenatide, Liraglutide, Lixisenatide, Albiglutide, Dulaglutide, Semaglutide and Taspoglutide.
  • the present invention provides a method of preparing a GLP-1 peptide, comprising the steps of: a. providing a first peptide fragment having amino acid sequence namely P -Arg (Pbf)-Gly-resin; b.
  • the side chain W attached to Lys 20 can be N-e(y Glu[N-a- hexadecanoyl]) for Liraglutide or AEEAc-AEEAc-y -Glul7- Carboxyheptadecanoyl for Semaglutide.
  • deprotecting agent which is a mixture of reagents selected from the group comprising of TFA, TIS, TIPS, DTT, EDT, ammonium iodide, 2,2'-(ethylenedioxy)diethane and acetyl cysteincysteine, DMS, phenol, cresol and thiocresol.
  • the coupling of peptide fragments and amino acids are carried out by using coupling agent, base and organic solvents.
  • the coupling agent used for coupling of peptide fragments are selected from the group comprising of hydroxybenzotriazole (HOBt); O-(7- azabenzotriazol- 1 -yl)- 1 , 1 ,3 ,3tetramethyluronium hexafluorophosphate (HATU), O-(benzotriazol-l-yl)-N,N,N',N'tetramethyluronium tetrafluoroborate (TBTU), 1,3-dicyclohexylcarbodlimide (DCC), l(dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC HC1), diisopropylcarbodiimide (DIC), isopropylchloroformate (IPCF), O- (benzotriazol- 1 -yl) 1 , 1 ,3 ,3-tetramethyluronium hexafluorophosphate
  • HBTU benzotriazol- l-yl-oxytris(dimethyl-amino)-phosphonium hexafluorophosphate
  • BOP-CI N,N-bis-(2-oxo-3-oxazolidinyl)phosphonic dichloride
  • BOP-CI benzotriazo lyloxytri(pyrolidino)phosphoniumhexafluorophosphate(PyB OP), bromotri(pyrrolidino)phosphonium hexafluorophosphate (PyBrOP), chlorotri(pynolidino)phosphonium hexafluorophosphate (PyClOP), ethyl-2-cyano-2(hydroxyimino) acetate (Oxyma Pure), O-(6-Chloro-l- hydrocibenzotriazol- 1 -yl)- 1 , 1 ,3 ,3tetramethyluronium tetrafluoroborate (TCTU),
  • the base used for coupling of peptide fragments are selected from a group comprising but not limiting to di-Cl-ClO -alkyl sulphides, alkyl phenyl sulphides, piperidine, 1,2-aminothiol of cysteine, a 1,2-aminoethanol of serine, a 1,2-aminoethanol of threonine, an aminooxyacetyl functional group, a mono-hydrazine succinyl functional group, ammonia, hydrazine, an alkoxide, a 4-hydrazinobenzoyl functional group, diisopropylamine, N,N-diisopropylethylamine triethylamine, dimethylamine, trimethyl amine, isopropyl ethylamine, pyridine, N- methyl morpholine and mixtures thereof.
  • the organic solvent used for coupling of peptide fragments are selected from a group comprising of DMF, DCM, THF, NMP, DMAC methanol, ethanol, isopropanol, dichloroethane, 1,4-dioxane, 2-methyl tetrahydrofuran ethyl acetate, acetonitrile, acetone.
  • the whole peptide sequence having amino acid sequence SEQ ID NO: 12 so obtained from the method of preparation of GLP-1 peptide is further deprotected followed by resin cleavage in presence of cleavage mixture of TFA: Phenol: DODT: Thioanisole: H2O having ratio of 82.5 : 5.0 : 2.5 : 5.0 : 5.0.
  • the resin cleaved peptide having amino acid sequence SEQ ID NO: 12 is further filtered to obtain the crude GLP-1 peptide.
  • the whole peptide sequence having amino acid sequence SEQ ID NO: 12 is Semaglutide.
  • a method of preparation of GLP- 1 peptide comprising one or more coupling steps with peptide fragments having amino acid SEQ ID NO: 1, 3 and Fragment BG with other amino acid fragments, wherein the steps comprises i. providing a first peptide fragment having amino acid sequence namely P -Arg (Pbf)-Gly-resin, ii. deprotecting said first peptide fragment in presence of a deprotecting agent and coupling the deprotected first peptide fragment with peptide fragment having amino acid sequence SEQ ID NO:1 to form peptide fragment having amino acid sequence SEQ ID NO:5 in presence of a coupling agent, a base and a solvent, iii.
  • a method for preparation of GLP-1 peptide comprising one or more coupling steps with peptide fragments having amino acid SEQ ID NO: l(Fragment IG), SEQ ID NO: 4 (Fragment YF)and Fragment BG with other amino acid fragments, wherein the steps comprises p. providing a first peptide fragment having amino acid sequence namely P -Arg (Pbf)-Gly-resin, q.
  • the method for preparation of GLP-1 peptide after the steps of coupling comprises the step of global deprotection of the long peptide sequence so formed by one or more coupling steps in presence of a cleavage mixture comprising TFA: Phenol: DODT: Thioanisole: H2O having ratio 82.5: 5.0: 2.5 : 5.0 : 5.0 to obtain the crude GLP-1 peptide which is further filtered to obtain the final pure GLP-1 peptide.
  • the crude GLP- 1 peptide as obtaining in accordance with the present invention is isolated by lyophilisation and is further purified by reverse phase HPLC using solvents.
  • the solvents used in reverse HPLC purification is selected from TFA in water, acetic acid, acetonitrile, orthophosphoric acid in water, triethylamine in water, ammonium acetate in water and ammonium bicarbonate in water.
  • the present method involving the coupling of one or more peptide fragments selected from the peptide fragment sequence SEQ ID NO: 1 (Fragment IG), SEQ ID NO: 2(Fragment SG), Fragment BG, SEQ ID NO: 3(Fragment YF) and SEQ ID NO: 4(Fragment YA) results in preparation of crude GLP-1 peptides, which excludes multiple steps of HPLC purification to obtain the pure peptide.
  • crude Semaglutide obtained by using the peptide fragments having amino acid sequences SEQ ID NO: l(Fragment IG), SEQ ID NO: 2(Fragment SG), Fragment BG, SEQ ID NO: 3(Fragment YF), and SEQ ID NO: 4(Fragment YA) involves HP 20 resin treatment followed by single step of HPEC, which results in formation of Semaglutide having >99.5% purity.
  • Example 1 Synthesis of Peptide Fragment having amino add SEQ ID NO: 1 (Fragment IG): Fmoc-Ile-Ala-Trp (Boc)-Leu-Val-Arg (Pbf)-Gly-OH:-
  • 2-CTC resin 250 g, loading capacity 1.51 mmol/g, 100 -200 mesh
  • Resin was swelled in DCM (10 Vol.) for 30 minutes, two times each.
  • Fmoc deprotection is performed by adding 20 % Piperidine in DMF (2 x 10 Vol.) for 10 minutes followed by 15minutes respectively. The solvent was drained and resin was washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.
  • Fmoc deprotection and coupling procedure were followed for following amino acids as per sequence of amino acids, Fmoc-Arg (Pbf)-OH, Fmoc-Val-OH (384.35 g, 3.0 eq), Fmoc-Leu-OH (400.22 g, 3.0 eq), Fmoc-Trp (Boc)-OH (596.37 g, 3.0 eq), Fmoc-Ala-OH (352.5 g, 3.0 eq), Fmoc-Ile-OH (400.33 g, 3.0 eq) were performed similar to the above procedure.
  • Resin cleavage Peptide resin was washed with DCM (1 x 10 Vol.) for 2-3 minutes and then filtered. Peptide was cleaved from the resin using 1% TFA/ DCM (3 x 10 Vol.), 5 minutes for each time. Cleavage fractions were collected and neutralized with pyridine (1:1 Volume ratio to TFA in each cleavage). Combined cleavage fractions were concentrated under reduced pressure at 30-35 °C. Resulting solution was co-evaporated with ethanol (2 x 5 Vol.) up to minimum volume and slurry was added to purified water (50 Vol.), stirred for 18h.
  • Example 2 Synthesis of Peptide Fragment having amino acid SEQ ID NO: 2 (Fragment SG): Fmoc-Ser (tBu) - Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH Peptide fragment having amino acid sequence SEQ ID NO: 2 was synthesized by using 2-CTC resin by Fmoc solid phase peptide strategy.
  • 2-CTC resin (50.0 g, loading capacity 1.7 mmol/g, 100 -200 mesh) was charged in a clean dry peptide synthesizer vessel. Resin was swelled by DCM (10 Vol.) for 30 minutes, 2 times each.
  • Fmoc deprotection was performed by adding 20 % piperidine in DMF (2 x 10 Vol.) for 10 minutes followed by 15minutes respectively. The solvent was drained and resin was washed by using DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.
  • Fmoc deprotection and coupling procedure were followed for following amino acids as per sequence of amino acids, Fmoc-Leu-OH (90.0 g, 3.0 eq), Fmoc- Tyr(tBu)-OH (117.1 g, 3.0 eq), Fmoc-Ser(tBu)-OH (97.76 g, 3.0 eq), Fmoc- Ser(tBu)-OH (97.6 g, 3.0 eq) were performed similar to the above procedure.
  • peptide resin was washed with 20 % Methanol in DCM (lx 10 Vol.), DCM (2 x 10 Vol.) and MTBE (2 x 10 Vol.) respectively.
  • Resin cleavage Peptide resin was washed with DCM (1 x 10 Vol) for 2-3 minutes and then filtered. Peptide fragment was cleaved from the resin using 1% TFA/ DCM (3 x 10 Vol.), 5 minutes for each time. Cleavage fractions were collected and neutralized with pyridine (1:1 Volume ratio to TFA in each cleavage), cleaved fractions were concentrated under reduced pressure at 30-35°C. Resulting solution was co-evaporated with ethanol (2 x 5 Vol.) up to minimum volume and slurry was added to purified water (50 Vol.), stirred for 18hours. Solid was filtered over Buchner funnel, washed with purified water (2 x5 Vol.).
  • Peptide fragment Fragment BG was synthesized on 2-CTC resin by Fmoc solid phase peptide strategy.
  • 2-CTC resin 250.0 g, loading capacity 1.8 mmol/g, 100 -200 mesh
  • Resin was swelled in DCM (lOVol.) for 30 minutes, 2 times each.
  • Fmoc deprotection was performed by adding 20 % Piperidine in DMF (2 x 10 Vol.) for 10 minutes followed by lOminutes respectively. The solvent was drained and, resin was washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.
  • Fmoc deprotection was performed by adding 20 % piperidine in DMF (2 x 10 Vol.) for 10 minutes followed by lOminutes respectively. The solvent was drained and resin was washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.
  • Resin cleavage Peptide Resin was washed with DCM (1 x 10 Vol.) for 2-3 minutes. Resin was cleaved by using 10 % TFE in DCM (8 x 10 Vol.), each wash was carried for 45 minutes. Combined fractions were concentrated under reduced pressure at 30-35°C. Resulting solution was co-evaporated with MTBE (2 x 5 Vol.), n-heptane (1 x 10 Vol.), dried under reduced pressure for 2-3 hours.
  • Example 4 Synthesis of Peptide Fragment having amino acid SEQ ID NO: 3 (Fragment YF):-Fmoc-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala- Lys(AEEAc-AEEAc-y-Glu-17-Carboxyheptadecanoyl)-Glu(OtBu)-Phe-OH:-
  • Peptide fragment having amino acid sequence SEQ ID NO: 3 was synthesized using 2-CTC resin by Fmoc solid phase peptide strategy.
  • 2-CTC resin (5.0 g, Loading capacity- 1.51 mmol/g) was charged in a clean dry peptide synthesizer vessel. Resin was swelled with DCM (10 Vol for30 minutes, 2 times each.
  • Fmoc deprotection was performed by adding 20 % Piperidine in DMF (2 x 10 Vol.) for 10 minutes followed by 15minutes respectively. The solvent was drained and resin was washed by DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.
  • Fmoc deprotection was performed by adding 20 % Piperidine in DMF (2 x 10 Vol.) for 10 minutes followed by 15minutes respectively. The solvent was drained and resin was washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.
  • Resin cleavage Peptide was cleaved from the resin using 1% TFA / DCM (3 x 10 Vol.), 5 minutes for each time. Cleavage fractions were collected and neutralized with pyridine (1:1 Volume ratio to TFA in each cleavage). Combined cleavage fractions were concentrated under reduced pressure at 30-35 °C. Resulting solution was co-evaporated with ethanol (2 x 5 Vol.) up to minimum volume and slurry was added to purified water (50 Vol.), stirred for 18hours. Solid was filtered over Buchner funnel, and washed with purified water (2 x 5 Vol.). Solid was washed with n-heptane (2 x 5 Vol.) and dried at 35-40 °C to afford white solid.
  • Example 5 Synthesis of Peptide Fragment having amino acid SEQ ID NO: 4 (Fragment YA): Fmoc-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-OH
  • Peptide fragment having amino acid sequence SEQ ID NO: 4 (Fragment YA) was synthesized on 2-CTC resin by Fmoc solid phase peptide strategy.
  • 2-CTC resin 250 g, loading capacity 1.8 mmol/g, 100 -200 mesh
  • Resin was swelled in DCM (lOVol.) for 30 min, two times each.
  • Fmoc deprotection was performed by adding 20 % Piperidine in DMF (2 x 10 Vol.) for 10 minutes followed by lOminutes respectively. The solvent was drained and resin was washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.
  • Fmoc deprotection and coupling procedure were followed for following amino acids as per sequence of amino acids, Fmoc-Gln(Trt)-OH (673.5 g, 3.0 eq), Fmoc- Gly-OH (401.35 g, 3.0 eq), Fmoc-Glu(OtBu)-OH (574.4 g, 3.0 eq), Fmoc-Leu-OH (477.05 g, 3.0 eq), Fmoc-Tyr(tBu)-OH (620.35 g, 3.0 eq) were performed similar to the above procedure.
  • Resin cleavage Peptide resin was washed with DCM (1 x 10 Vol.) for 2-3 minutes. Peptide product was cleaved from resin using 1% TFA in DCM (3 x 10 Vol.), 5 minutes for each time. Cleaved fractions were collected and neutralized with pyridine (1:1 Volume ratio to TFA for each cleavage). Combined cleavage fractions were concentrated under reduced pressure at 30-35°C. Resulting solution was coevaporated with ethanol (2 x 5 Vol.) up to minimum volume and slurry was added to purified water (50 Vol.), stirred for 18 hours.
  • Fmoc deprotection was performed by adding 20 % Piperidine in DMF (2 x 10 Vol.) for 10 minutes followed by 15 minutes respectively. The solvent was drained and resin was washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.
  • Fmoc deprotection was performed by adding 20 % Piperidine in DMF (2 x 10 Vol.) for 10 minutes followed by 15minutes respectively. The solvent was drained and washed the resin with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.
  • Fmoc deprotection was performed by adding 20 % Piperidine in DMF (2 x 10 Vol.) for 10 minutes followed by 15minutes respectively. The solvent was drained and resin was washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.
  • Fmoc deprotection was performed by adding 20% piperidine in DMF (2 x 10 Vol.) for 10 minutes followed by 15 minutes respectively. The solvent was drained and resin was washed with DMF (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.
  • Fmoc-Ser(tBu)-OH coupling - The pre-activated Fmoc-Ser(tBu)-OH (1.72 g, 3.0 eq) with HOBt monohydrate (0.69 g, 3.0 eq) and DIC (0.70 mL, 3.0 eq) in DMF (10 Vol.) was added to above resin. Coupling continued for 6 hours. Solvent was drained and resin was washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 xlO Vol.) respectively.
  • Fmoc deprotection and coupling procedure were followed for following amino acids as per sequence of amino acid, Fmoc- Ser(tBu)-OH (1.72 g, 3.0 eq), Fmoc- Val-OH (1.52 g, 3.0 eq), Fmoc- Asp(OtBu)-OH (1.92 g, 3.0 eq), Fmoc-Ser(tBu)- OH (1.72 g, 3.0 eq), Fmoc- Thr(tBu)-OH (1.78 g, 3.0 eq), Fmoc-Phe-OH (1.74 g, 3.0 eq), Fmoc-Thr(tBu)-OH (1.78 g, 3.0 eq) were performed similar to the above procedure.
  • Fmoc deprotection was performed by adding 20 % Piperidine in DMF (2 x 10 Vol.) for 10 minute followed by 15minute respectively. Drained the solvent, washed the resin with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.
  • Fmoc deprotection was performed by adding 20 % piperidine in DMF (2 x 10 Vol.) for 10 minutes followed by 15 minutes respectively. Drained the solvent, washed the resin with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.
  • Boc-His(trt)-OH Boc-His(Boc)-OH was also used.
  • peptide resin was washed with 20 % Methanol in DCM (2 x lOVol.) and MTBE (1 x 10 Vol.) to afford dried peptide resin (12.2 g).
  • HP 20 resin treatment HP20 resin (120.0 g) is swelled with Methanol (14 Vol.) for 10 min, filtered, washed with purified water (3 x 14 Vol.). Crude Semaglutide (6 g, assay 44.62%) was dissolved in 0.25M Ammonium bicarbonate (200 Vol.) and loaded to above resin, agitated for 2 hrs. After 2 hrs, resin was loaded in glass column and washed with H2O (3 x 14 Vol.) followed by 2.5, 5.0, 7.5, 10.0 and 40.0 of ACN/H2O. Fractions were collected and lyophilized to afford off white solid (4.1 g, HPLC purity 63.4 %, 58.7 % assay).
  • impure Semaglutide was further purified by preparative RP-HPLC. Desired purity fractions were pooled and lyophilized to afford white solid (1.34 g, HPLC Purity 99.52 %).
  • Example 7 Preparation of Semaglutide involving coupling of peptide fragments having amino acid sequences SEQ ID NO: 1 (Fragment IG), SEQ ID NO: 2 (Fragment SG), (Fragment BG) and amino acids
  • Fmoc-Phe-OH coupling - The pre-activated Fmoc-Phe-OH(1.74 g, 3.0 eq) with HOBt monohydrate(0.69 g, 3.0 eq) and DIC (0.70 mL, 3.0 eq) in DMF (10 Vol.) added to above resin. Coupling was continued for 6 hours. Solvent was drained and peptide resin was washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 xlO Vol.) respectively. Fmoc deprotection and coupling procedure were followed for following amino acid as per sequence which are Fmoc-Glu (OtBu)-OH (1.91 g, 3.0 eq), were performed similar to the above procedure.
  • Fmoc deprotection Fmoc deprotection performed by adding 20 % Piperidine in DMF (2 x 10 Vol.) for 10 minutes followed by 15minutes respectively. Drained the solvent, washed the resin with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.
  • Fmoc deprotection and coupling procedure were followed for following amino acid as per sequence, which are, Fmoc-Ala-OH (1.40 g, 3.0 eq), Fmoc-Ala-OH (1.40 g, 3.0 eq), Fmoc-Gln(Trt)-OH (2.77 g, 3.0 eq) were performed similar to the above Fmoc-Phe-OH coupling procedure.
  • Fmoc deprotection was performed by adding 20% Piperidine in DMF (2 x 10 Vol.) for 10 min. followed by 15 min respectively. Drained the solvent, washed resin with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.
  • Fmoc deprotection and coupling procedure were followed for following amino acids as per sequential, which are Fmoc-Asp (OtBu)-OH (1.85 g, 3.0 eq), Fmoc- Ser(tBu)-OH (1.72 g, 3.0 eq), Fmoc- Thr(tBu)-OH (1.78 g, 3.0 eq), Fmoc-Phe-OH (1.74 g, 3.0 eq), Fmoc-Thr(tBu)-OH (1.78 g, 3.0 eq), were performed similar to the above procedure.
  • Fmoc deprotection Fmoc deprotection performed by adding 20 % Piperidine in DMF (2 x 10 Vol.) for 10 minutes followed by 15minutes respectively. Drained the solvent, washed the resin with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.
  • peptide fragment Fragment BG coupling - The pre-activated solution of peptide fragment Fragment BG, Fmoc-Aib-Glu(OtBu)-Gly-OH (2.55 g, 3.0 eq) with HBTU(1.7 g, 3.0 eq) and HOBt monohydrate (0.69 g, 3.0 eq) and DIPEA (0.78, 3.0 eq) in 20% DMSO/DMF (10 Vol.) added to above peptide resin, agitated for 5h. Drained solvent and resin washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.), DMF (2 x 10 Vol.) respectively.
  • Fmoc deprotection was performed by adding 20% Piperidine in DMF (2 x 10 Vol.) for 10 minutes, followed by 15 minutes respectively. Drained the solvent, washed the resin with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.
  • Boc-His(Boc)-OH coupling - The pre-activated solution of Boc-His(Boc)-OH (2.23 g, 3.0 eq) with HBTU(1.7 g, 3.0 eq), HOBt monohydrate (0.69 g, 3.0 eq) and DIPEA (0.78 mL, 3.0 eq) in DMF (10 Vol.) added to above peptide resin. Resin was agitated for 4h. Drained solvent and resin washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.), DMF (2 x 10 Vol.) respectively.
  • peptide resin was washed with 20% Methanol in DCM (2 x lOVol.), MTBE (1 x 10 Vol) and well dried to afford 12.0 g of peptide resin.
  • HP 20 resin treatment HP20 resin (100.0 g) is swelled with Methanol (14 Vol.) for 10 min, filtered, washed with purified water (3 x 14 Vol.). Crude Semaglutide (5 g, purity 61.2%) was dissolved in 0.25M Ammonium bicarbonate (200 Vol.) and loaded to above resin, agitated for 2 hrs. After 2 hrs, resin was loaded in glass column and washed with H2O (3 x 14 Vol.) followed by 2.5, 5.0, 7.5, 10.0 and 40.0 of ACN/H2O. Fractions were collected and lyophilized to afford off white solid (3.3 g)-
  • impure Semaglutide was further purified by preparative RP-HPFC. Desired purity fractions were pooled and lyophilized to afford white solid (0.91 g, HPLC Purity 99.63 %).
  • Example 8 Preparation of Semaglutide involving coupling of peptide fragments having amino acid sequences SEQ ID NO: 1 (Fragment IG), SEQ ID NO:4 (Fragment YA), Fragment BG and amino acids
  • Fmoc deprotection was performed by adding 20 % Piperidine in DMF (2 x 10 Vol.) for 10 minutes followed by 15minutes respectively. Drained the solvent, washed the resin with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.
  • Fmoc-Phe-OH coupling The pre-activated solution of Fmoc-Phe-OH(1.74 g, 3.0 eq) with HOBt monohydrate(0.69 g, 3.0 eq) and DIC (0.70 mF, 3.0 eq) in DMF (10 Vol.), added to Fmoc-deprotected resin and agitated for 5 hours. After 5 hours solvent was drained and resin was washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.), DMF (2 x 10 Vol.) respectively.
  • Fmoc deprotection Fmoc deprotection performed by adding 20 % Piperidine in DMF (2 x 10 Vol.) for 10 minutes followed by 15minutes respectively. Drained the solvent, washed the resin with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.
  • Fmoc deprotection was performed by adding 20 % piperidine in DMF (2 x 10 Vol.) for 10 minutes followed by 15minutes respectively. Drained the solvent, washed the resin with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.
  • Fmoc deprotection Fmoc deprotection performed by adding 20% piperidine in DMF (2 x 10 Vol.) for 10 minutes followed by 15 minutes respectively. Drained the solvent, washed resin with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.
  • Fmoc-Ser (tBu)-OH of coupling The pre-activated solution of Fmoc-Ser (tBu)-OH (1.72 g, 3.0 eq) with HOBt monohydrate (0.69 g, 3.0 eq) and DIC (0.70 mL, 3.0 eq) in DMF (10 Vol.), was added to resin and agitated for 5 hrs. After 5 hrs, solvent drained and resin was washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.), DMF (2 x 10 Vol.) respectively.
  • Fmoc deprotection and coupling procedure were followed for following amino acids as per sequential. Which are Fmoc-Ser(tBu)-OH (1.72 g, 3.0 eq), Fmoc-Val- OH (1.52 g, 3.0 eq), Fmoc-Asp(OtBu)-OH (1.85 g, 3.0 eq), Fmoc-Ser(tBu)-OH (1.72 g, 3.0 eq), Fmoc- Thr(tBu)-OH (1.78 g, 3.0 eq), Fmoc-Phe-OH (1.74 g, 3.0 eq), Fmoc-Thr(tBu)-OH (1.78 g, 3.0 eq) were performed similar to the above procedure.
  • Fmoc deprotection Fmoc deprotection performed by adding 20 % piperidine in DMF (2 x 10 Vol.) for 10 minutes followed by 15minutes respectively. Drained the solvent, washed resin with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.
  • peptide fragment Fragment BG, Fmoc-Aib-Glu (OtBu)-Gly-OH coupling - The pre-activated solution of peptide fragment Fragment BG, Fmoc-Aib-Glu(OtBu)- Gly-OH (2.55 g, 3.0 eq) with HBTU (1.7 g, 3.0eq) HOBt monohydrate(0.69 g, 3.0 eq) and DIPEA (0.78 mL, 3.0eq) in 20 % DMSO/DMF (10 Vol.), DIC (0.70 mL, 3.0 eq) was added to resin and agitated for 5 hrs. After 5 hours, solvent was drained and peptide resin was washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.), DMF (2 x 10 Vol.) respectively.
  • Fmoc deprotection Fmoc deprotection performed by adding 20% Piperidine in DMF (2 x 10 Vol.) for 10 minutes followed by 15 minutes respectively. Drained the solvent, washed resin with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.
  • Boc-His(Trt)-OH coupling The pre-activated solution of Boc-His (Trt)-OH (2.41 g, 3.0 eq) with HBTU (1.7 g, 3.0eq) HOBt monohydrate(0.69 g, 3.0 eq) and DIPEA (0.78 mL, 3.0eq) in 20 % DMSO/DMF (10 Vol.), DIC (0.70 mL, 3.0 eq) added to resin and agitated for 5 hrs. After 5 hrs, solvent drained and peptide resin washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.), DMF (2 x 10 Vol.) respectively.
  • peptide resin was washed with 20% Methanol in DCM (2 x lOVol.), MTBE (1 x 10 Vol) and dried well to afford 13.25 g of peptide resin.

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Abstract

La présente invention concerne un procédé de préparation de peptides GLP-1 à l'aide de différents fragments peptidiques ayant des séquences d'acides aminés SEQ ID NO : 1 (fragment IG), SEQ ID NO : 2 (fragment SG), fragment BG, SEQ ID NO : 3 et SEQ ID NO : 4 5 et des combinaisons de ceux-ci. Le procédé de préparation est sans danger pour l'environnement, simple, facile et efficace et fournit le produit final avec un haut rendement et une pureté élevée. L'invention concerne également le procédé de purification des peptides.
PCT/IB2022/062039 2021-12-10 2022-12-12 Synthèse d'analogues de glp-1 WO2023105497A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180057558A1 (en) * 2014-09-23 2018-03-01 Novetide, Ltd. Synthesis of GLP-1 Peptides
WO2019069274A1 (fr) * 2017-10-04 2019-04-11 Chemical & Biopharmaceutical Laboratories Of Patras S.A. Procédé de préparation d'un peptide de type glucagon
CN111944039A (zh) * 2019-04-30 2020-11-17 深圳市健元医药科技有限公司 一种索玛鲁肽的合成方法
CN112028986A (zh) * 2020-09-11 2020-12-04 哈尔滨吉象隆生物技术有限公司 一种司美格鲁肽的合成方法
CN113444164A (zh) * 2021-06-29 2021-09-28 台州吉诺生物科技有限公司 一种固相合成索马鲁肽的方法
WO2021224938A1 (fr) * 2020-05-05 2021-11-11 Neuland Laboratories Limited Procédé amélioré pour la préparation de sémaglutide

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180057558A1 (en) * 2014-09-23 2018-03-01 Novetide, Ltd. Synthesis of GLP-1 Peptides
WO2019069274A1 (fr) * 2017-10-04 2019-04-11 Chemical & Biopharmaceutical Laboratories Of Patras S.A. Procédé de préparation d'un peptide de type glucagon
CN111944039A (zh) * 2019-04-30 2020-11-17 深圳市健元医药科技有限公司 一种索玛鲁肽的合成方法
WO2021224938A1 (fr) * 2020-05-05 2021-11-11 Neuland Laboratories Limited Procédé amélioré pour la préparation de sémaglutide
CN112028986A (zh) * 2020-09-11 2020-12-04 哈尔滨吉象隆生物技术有限公司 一种司美格鲁肽的合成方法
CN113444164A (zh) * 2021-06-29 2021-09-28 台州吉诺生物科技有限公司 一种固相合成索马鲁肽的方法

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