An improved process for the preparation of tirzepatide by using hybrid approach

Abstract

An improved process for the preparation of Tirzepatide or a pharmaceutically acceptable salt thereof, by using a hybrid approach having the chemical structure of Formula I. comprising condensation of Fmoc-T(tBu)-F-T(tBu)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I- Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(Linker)-OH (Fragment II) with H-A-F-V- Q(Trt)-W(Boc)-L-I-A-G-G-P-S(tBu)-S(tBu)-G-A-P-P-P-S(tBu)-NH2 (Fragment III) in the presence of coupling agent and solvent to obtain H-T(tBu)-F-T(tBu)-S(tBu)- D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(Linker)-A-F-V- Q(Trt)-W(Boc)-L-I-A-G-G-P-S(tBu)-S(tBu)-G-A-P-P-P-S(tBu)-NH2 compound of Formula II, which is on coupling with Fmoc-Y(tBu)-Aib-E(OtBu)-G-OH (Fragment I) to obtain Tirzepatide. The present invention also provides novel fragments I, II & III and their processes for preparation, which are useful in the manufacturing of Tirzepatide or a pharmaceutically acceptable salt thereof.

Description

[0001] AN IMPROVED PROCESS FOR THE PREPARATION OF TIRZEPATIDE BY USING HYBRID APPROACH

[0002] Related Patent Application(s): This application claims priority and to benefits from Indian provisional patent application No. 202441051183 filed on July 04, 2024; the disclosure of which is incorporated herein by reference.

[0003] Field of the Invention The present invention relates to an improved process for the preparation of Tirzepatide or a pharmaceutically acceptable salt thereof by using a hybrid approach, having the chemical structure of Formula I.

[0004] Formula I The present invention also provides a novel Fragments I, II & III and their processes of preparation, which are useful in the manufacturing of Tirzepatide, or a pharmaceutically acceptable salt thereof.

[0005] Fmoc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-OH

[0006] Fragment I Background of the Invention

[0007] Tirzepatide is chemically known as L-Tyrosyl-2-methylalanyl-L-a-glutamylglycyl-L- threonyl-L-phenylalanyl-L-threonyl-L-seryl-L-a-aspartyl-L-tyrosyl-L-seryl-L-isoleucyl- 2-methylalanyl-L-leucyl-L-a-aspartyl-L-lysyl-L-isoleucyl-L-alanyl-L-glutaminyl-N6- [(22S)-22,42-dicarboxy-l,10,19,24-tetraoxo-3,6,12,15-tetraoxa- 9, 18, 23- triazadotetracont-l-yl]-L-lysyl-L-alanyl-L-phenylalanyl-L-valyl-L-glutaminyl-L- tryptophyl-L-leucyl-L-isoleucyl-L-alanylglycylglycyl-L-prolyl-L-seryl-L-serylglycyl-L- alanyl-L-prolyl-L-prolyl-L-prolyl-L-serinamide, which is shown as in three-letter code H-Tyr-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-Aib-Leu-Asp-Lys-Ile- Ala-Gin- Lys(Linker)-Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro- Pro-Ser-NHi. The molecular formula is C225H348N48O68. The structural formula is:

[0008] Formula I

[0009] Tirzepatide is a linear polypeptide of 39 amino acids which has been chemically modified by lipidation to improve its uptake into cells and its stability to metabolism, whose amino acid residues contains 2 non-coded amino acids (aminoisobutyric acid, Aib) in positions 2 and 13, a C-terminal amide, and Lys residue at position 20 that is attached to 1, 20- eicosanedioic acid via a linker which consists of a Glu and two 8-amino-3,6- dioxaoctanoic acids.

[0010] Tirzepatide is a first-in-class medication that activates both the GIP (gastric inhibitory polypeptide) and GLP-1 (glucagon-like peptide- 1) dual receptor agonist targeted as a treatment for diabetes as well as non-alcoholic steatohepatitis (NASH) and chronic weight management.

[0011] Tirzepatide is first disclosed in US 9474780 B2, and the disclosed process leads to the formation of impurities and hence additional purification techniques are required to get pure Tirzepatide. The disclosed process is highly expensive and commercially not viable. Several processes for preparation of Tirzepatide and its fragments have been disclosed in the literature; WO 2024112617 A2, IN 202241061443 A, IN202241049251 A, WO 2020 / 159949 Al, WO 2021 / 158444 Al, CN 112110981 A, CN 112661815 A, WO 2022079639 Al and WO 2021260530 Al.

[0012] From the foregoing, it is apparent that the reported methods for the preparation of Tirzepatide requires stringent operational conditions, which are not only tedious but also results in significant yield loss. The processes require longer reaction time for the completion at several stages including tedious work up procedures and purification steps.

[0013] In view of the above, there is a significant need to develop an improved technology for production of Tirzepatide having a combination of advantages including commercially desired purity and efficiency. Similarly, there is a need for efficient and environmentally green process, including stable intermediates to provide Tirzepatide of Formula I with less purification steps, high purity and good yield.

[0014] Summary of the Invention

[0015] The present invention provides an improved process for the preparation of Tirzepatide or a pharmaceutically acceptable salt thereof, using fragments through hybrid approach.

[0016] The present invention provides a cost effective, novel and an efficient process for the preparation of Tirzepatide, producing minimal waste streams for both environmental and enhanced operational safety, its intermediates by making appropriate fragments in a solid phase approach, followed by condensing these fragments by using solution phase approach with higher yields and purity.

[0017] The present invention further involves the coupling of appropriate fragments which are synthesised on solid support 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 Tirzepatide or a pharmaceutically acceptable salt thereof. In the first aspect, the present invention relates to a process for the preparation of Tirzepatide compound of Formula I or a pharmaceutically acceptable salt thereof using a hybrid approach,

[0018] Formula I which comprises: i) condensation of Fmoc-T(tBu)-F-T(tBu)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib- L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(Linker)-OH (Fragment II) with H-A-F-V- Q(Trt)-W(Boc)-L-I-A-G-G-P-S(tBu)-S(tBu)-G-A-P-P-P-S(tBu)-NH2(Fragment III) in the presence of coupling agent and solvent, followed by in-situ manner deprotection in the presence of base to obtain H-T(tBu)-F-T(tBu)-S(tBu)- D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(Linker)-A-F- V-Q(Trt)-W(Boc)-L-I-A-G-G-P-S(tBu)-S(tBu)-G-A-P-P-P-S(tBu)-NH2compound of Formula II; ii) condensing Fmoc-Y(tBu)-Aib-E(OtBu)-G-OH (Fragment I) with H-T(tBu)-F- T(tBu)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)- K(Linker)-A-F-V-Q(Trt)-W(Boc)-L-I-A-G-G-P-S(tBu)-S(tBu)-G-A-P-P-P- S(tBu)-NH2compound of Formula II in the presence of coupling agent and solvent, followed by in-situ manner deprotection in the presence of base to obtain H-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(tBu)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I- Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(Linker)-A-F-V-Q(Trt)-W(Boc)-L-I-A-G- G-P-S(tBu)-S(tBu)-G-A-P-P-P-S(tBu)-NH2compound of Formula III; iii) global deprotection of compound of Formula III using a reagent and solvent to obtain crude Tirzepatide or a pharmaceutically acceptable salt thereof; iv) purifying the crude Tirzepatide by preparative HPLC to obtain pure Tirzepatide or a pharmaceutically acceptable salt thereof. The present invention provides a Fragment I: Fmoc-Y(tBu)-Aib-E(OtBu)-G-OH.

[0019] The present invention provides a Fragment II: Fmoc-T(tBu)-F-T(tBu)-S(tBu)-D(OtBu)- Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(Linker)-OH.

[0020] The present invention provides a Fragment III: H-A-F-V-Q(Trt)-W(Boc)-L-I-A-G-G-P- S(tBu)-S(tBu)-G-A-P-P-P-S(tBu)-NH2.

[0021] Detailed Description of the Invention

[0022] The present invention provides an improved process for the preparation of Tirzepatide or a pharmaceutically acceptable salt thereof by making appropriate fragments on solid support, followed by condensing these fragments using solution phase approach which provides a desired product with higher yields and purity.

[0023] The improved process as described in the present invention involves the use of novel fragments, also provides various embodiments for the preparation of intermediates, which are useful for the production of Tirzepatide are depicted as follows:

[0024] Fragment I: Fmoc-Y(tBu)-Aib-E(OtBu)-G-OH.

[0025] Fragment II: Fmoc-T(tBu)-F-T(tBu)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)- K(Boc)-I-A-Q(Trt)-K(Linker)-OH

[0026] Fragment III: H-A-F-V-Q(Trt)-W(Boc)-L-I-A-G-G-P-S(tBu)-S(tBu)-G-A-P-P-P- S(tBu)-NH2.

[0027] Formula II: H-T(tBu)-F-T(tBu)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)- K(Boc)-I-A-Q(Trt)-K(Linker)-A-F-V-Q(Trt)-W(Boc)-L-I-A-G-G-P-S(tBu)-S(tBu)-G- A-P-P-P-S(tBu)-NH2

[0028] In an embodiment, the peptide fragments are prepared by using solid-phase peptide synthesis through a linear approach.

[0029] The three-letter amino acid abbreviations and the codes for other reagents used throughout the present invention are defined as follows: Alanine: (Ala) A Arginine: (Arg) R

[0030] Asparagine: (Asn) N Aspartic acid: (Asp) D

[0031] Cysteine: (Cys) C Glutamine: (Gin) Q

[0032] Glutamic acid: (Glu) E Glycine: (Gly) G

[0033] Histidine: (His) H Isoleucine: (He) I

[0034] Leucine: (Leu) L Lysine: (Lys) K

[0035] Methionine: (Met) M Phenylalanine: (Phe) F

[0036] Proline: (Pro) P Serine: (Ser) S

[0037] Threonine: (Thr) T Tryptophan: (Tip) W

[0038] Tyrosine: (Tyr) Y Valine: (Vai) V

[0039] 9-Fluorenylmethoxycarbonyl: Fmoc Di tert-butyl dicarbonate: Boc Trityl chloride: Trt Tert-butyl: tBu

[0040] Carboxybenzyl: Cbz Tert-butyl ester: OtBu

[0041] 2-Aminoisobutyric acid: Aib Pseudoproline dipeptide Oxa

[0042] 2-[2-(2-amino-ethoxy)- AEEA a-methyl leucine aMeL ethoxy] -acetyl,

[0043] Solid phase peptide synthesis is carried out on an insoluble polymer which is acid sensitive. Acid sensitive resin selected from the group consisting of 2-chloro trityl resin (CTC), wang resin, 4-methyltrityl chloride, sieber amide resin and rink acid resin. Preferably using 2-CTC resin and sieber amide resin. The resin used for the synthesis of Tirzepatide undergoes swelling in presence of a solvent selected from the group consisting of dichloromethane (MDC), N, A-Di methyl formamide (DMF) and N- methyl - 2-pyrrolidone (NMP) or its mixture. Solvents are used throughout the invention selected from the group consisting of hydrocarbon solvents such as dimethylacetamide, dimethylformamide (DMF), formamide, A-Methy 1 formamide, N- Methyl pyrrolidine (NMP), dimethyl acetamide (DMAC), methanol, ethanol, isopropanol, tert-butanol, dichloromethane, dichloroethane, 1,4-dioxane, di-isopropyl ether, diethyl ether, tetrahydrofuran, methyl tert-butyl ether, ethyl-tert-butyl ether, ethyl acetate, isopropyl acetate, n-butyl acetate, isobutyl acetate, methyl acetate, acetonitrile, propionitrile, butyronitrile, isobutyronitrile, acetone, ethyl methyl ketone, methyl isobutyl ketone, diethyl ketone, pentane, n-hexane, n-heptane, water or a mixture thereof.

[0044] The coupling agent used throughout the reaction may be selected from the group consisting of Ethylcyano (hydroxyimino)acetate-2-tri-(l-pyrrolidinyl)-Phosphonium hexa fluorophosphate (PyOxim), ethyl-2-cyano-2-(hydroxy amino) acetate (Oxyma pure), O-(benzotriazol-l-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), diisopropyl carbodiimide (DIC), 1,3-dicyclohexylcabodiimide (DCC), O-(7- azabenzotriazol-l-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), 1- (dimethyl aminopropyl)-3-ethylcarbodiimide hydrochloride (EDC HC1), O- (benzo triazol- 1-yl)- 1,1, 3, 3 -tetra methyluronium hexafluorophosphate (HBTU), 1- Hydroxybenzotriazole (HOBt), Isopropyl chloro formate (IPCF), Benzotriazol- 1-yl-oxy- tris(dimethyl-amino)-phosphonium hexa fluorophosphate (BOP), benzotriazole- 1- yloxytri(pyrrolidino)phosphonium hexa fluoro phosphate (PyBOP), N,N-bis-(2-oxo-3- oxazolidinyl)phosphonic dichloride (BOP-CI), bromotri(pyrrolidino)phosphonium hexa fluoro phosphate (PyBrOP), O-(6-Chloro-l-hydrocibenzotriazol-l-yl)-l, 1,3,3- tetramethyl uranium tetra fluoroborate (TCTU), chlorotri (pyrrolidino)phosphonium hexafluorophosphate (PyClOP), Ethyl l,2-dihydro-2-ethoxyquinoline- carboxylate(EEDQ), isobutyl chloro formate (IBCF), 2-succinimido-l, 1,3,3- tetramethyluronium tetrafluoroborate(TSTU), l-Cyano-2-ethoxy-2-oxo ethylidene aminooxy) dimethyl amino morpholino-carbeniumhexafluorophosphate (COMU), 2-(5- norbornen-2,3-dicarboximido)- 1 , 1 ,3,3-tetramethyluronium tetrafluoroborate (TNTU), propane phosphonic acid anhydride (PPAA), 3-(diethoxy phosphoryloxy)- 1,2,3- benzotriazin-4(3H)-one (DEPBT) or mixture thereof.

[0045] The base used throughout the reaction may be selected from the group consisting of 5- 10% palladium on carbon, 2-10% hydrazine hydrate, triethylamine, diisopropyl ethyl amine, A-methyl morpholine or pyridine.

[0046] An “isolated” peptide, as used herein, means a naturally-occurring peptide that has been separated or substantially separated from the cellular components (e.g., nucleic acids and other peptides) that naturally accompany it by purification, recombinant synthesis, or chemical synthesis, and also encompasses non-naturally-occurring recombinantly or chemically synthesized peptides that have been purified or substantially purified from cellular components, biological materials, chemical precursors, or other chemicals.

[0047] According to the present invention, 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 trifluoro acetic acid (TFA). The scavengers used are selected from the group consisting of TIPS, phenol, thioanisole, water or mixture thereof. Preferably the cocktail mixture used in the present invention is a mixture of TFA, TIPS, water and DTT in a preferred ratio of 90%: 5%: 5%: and 2.5%.

[0048] The protected amino acids used in the present invention are commercially available or may be prepared according to the procedures known in the literature.

[0049] In one embodiment, the process of the present invention comprises coupling of said polypeptides or pharmaceutically acceptable salts thereof, using solid phase peptide synthesis (SPPS), liquid phase peptide synthesis (LPPS) or a hybrid SPPS / LPPS approach.

[0050] The coupling reactions may be monitored by kaiser test, ninhydrin test, chloranil or TNBS test. The cleavage of the peptide from the solid support may be accomplished by any conventional methods which are well known in the art.

[0051] In one embodiment, the present invention relates to an improved process for the preparation of Tirzepatide or a pharmaceutically acceptable salt thereof by coupling appropriate fragments in a required sequence, deprotection and condensing them in solution phase, followed by purification to get Tirzepatide or a pharmaceutically acceptable salt thereof. The schematic description of the process of present invention is as shown in Scheme-I. Fmoc-T(tBu)-F-T(tBu)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(Linker)-OH

[0052] Fragment-II

[0053] EDC.HC1, H-A-F-V-Q(Trt)-W(Boc)-L-I-A-G-G-P-S(tBu)-S(tBu)- HOBt in DMF G-A-P-P-P-S(tBu)-NH2

[0054] Fragment-Ill

[0055] Step - 1

[0056] Fmoc-T(tBu)-F-T(tBu)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(Linker)-

[0057] H-A-F-V-Q(Trt)-W(Boc)-L-I-A-G-G-P-S(tBu)-S(tBu)-G-A-P-P-P-S(tBu)-NH2

[0058] De-Fmoc Water, n-Heptane, t-Butylamine, Methanol n-Heptane, DMF

[0059] H-T(tBu)-F-T(tBu)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu) (Linker)-

[0060] H-A-F-V-Q(Trt)-W(Boc)-L-I-A-G-G-P-S(tBu)-S(tB

[0061] Formula

[0062] EDC.HC1, Fmoc-Y(tBu)-X-E(OtBu)-G-OH

[0063] HOBt in DMF Fragment-I

[0064] Step - II

[0065] Fmoc-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(tBu)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)- I-A-Q(Trt)-K(AEEAc- AEEAc-y-Glu- 19-carboxynonadecanoyl mono-t-butyl ester)-A-F -V-Q(Trt)- W (Boc)- L-I-A-G-G-P-S(tBu)-S(tBu)-G-A-P-P-P-S(tBu)-NH2

[0066] Formula III

[0067] De-Fmoc

[0068] Water, n-Heptane,

[0069] Step - III t-Butylamine, Methanol n-Heptane, DMF Cleavage of protecting groups

[0070] Y-X-E-G-T-F-T-S-D-Y-S-I-X-L-D-K-I-A -Q-K(AEEAc-AEEAc-y-Glu- 19-carboxynonadecanoyl)-

[0071] A-F-V-Q-W-L-I-A-G-G-P-S-S-G-A-P-P-P-S-NH2

[0072] Tirzepatide Crude

[0073] „ Purification,

[0074] Stepr- IVTLyop ,hirlizat .i.on

[0075] Y-X-E-G-T-F-T-S-D-Y-S-I-X-L-D-K-I-A -Q-K(AEEAc-AEEAc-y-Glu-19-carboxynonadecanoyl)-

[0076] A-F-V-Q-W-L-I-A-G-G-P-S-S-G-A-P-P-P-S-NH2[Tirzepatide Pure]

[0077] Formula I

[0078] Scheme I

[0079] In step i), condensation of Fmoc-T(tBu)-F-T(tBu)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib- L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(Linker)-OH (Fragment II) with H-A-F-V-Q(Trt)- W(Boc)-L-I-A-G-G-P-S(tBu)-S(tBu)-G-A-P-P-P-S(tBu)-NH2(Fragment III) in the presence of coupling agent and solvent, followed by in-situ manner deprotection in the presence of base to obtain H-T(tBu)-F-T(tBu)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L- D(OtBu)-K(Boc)-I-A-Q(Trt)-K(Linker)-A-F-V-Q(Trt)-W(Boc)-L-I-A-G-G-P-S(tBu)- S(tBu)-G-A-P-P-P-S(tBu)-NH2 compound of Formula II.

[0080] In step i), the reaction temperature may range from 20 °C to 35 °C, preferably at a temperature in the range from 25°C to 30 °C. The duration of the reaction may range from 6 hours to 7 hours, preferably for a period of 6 hours.

[0081] In step ii), condensation of Fmoc-Y(tBu)-Aib-E(OtBu)-G-OH (Fragment I) with H- T(tBu)-F-T(tBu)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)- K(Linker)-A-F-V-Q(Trt)-W(Boc)-L-I-A-G-G-P-S(tBu)-S(tBu)-G-A-P-P-P-S(tBu)-NH2compound of Formula II in the presence of coupling agent and solvent followed by in- situ manner deprotection in the presence of base to obtain H-Y(tBu)-Aib-E(OtBu)-G- T(tBu)-F-T(tBu)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)- K(Linker)-A-F-V-Q(Trt)-W(Boc)-L-I-A-G-G-P-S(tBu)-S(tBu)-G-A-P-P-P-S(tBu)-NH2compound of Formula III.

[0082] In step ii), the reaction temperature may range from 20 °C to 35 °C, preferably at a temperature in the range from 25 °C to 30 °C. The duration of the reaction may range from 3 hour to 8 hours, preferably for a period of 7 hours.

[0083] In step iii), the global deprotection of compound of Formula III using a reagent and solvent to obtain crude Tirzepatide.

[0084] In step iii), the reaction temperature may range from 20 °C to 35 °C, preferably at a temperature in the range from 25 °C to 30 °C. The duration of the reaction may range from 3 hour to 7 hours, preferably for a period of 6 hours. In a preferred embodiment, the cleavage is subjected with a cocktail mixture consisting of TFA / TIPS / Water / DTT in the ratio ranges from 70% / 2.5% / 2.5% / l% to 95% / 10% / 10% / 5%, preferably the ratio of cocktail mixture is 90% / 5% / 5% / 2.5%. In step iv) purifying the crude Tirzepatide by preparative HPLC to obtain pure Tirzepatide or a pharmaceutically acceptable salt thereof.

[0085] Preparative HPLC method for purification of Tirzepatide:

[0086] Trifluoroacetic acid purification:

[0087] Column: UNISIL C18 (300x50 mm, 10 pm)

[0088] Sample preparation: 2.0 grams of crude Tirzepatide was dissolved in 200 mL of 50mM Ammonium bicarbonate in 1.0 L water solution. Filter the contents through 0.45p filter paper.

[0089] Mobile phase A: Trifluoro acetic acid (0.005 L) + water (5.0 L).

[0090] Mobile phase B: Acetonitrile (4.0 L) + water (5.0 L) + Trifluoro acetic acid (0.005 L) Equilibrate the column with 40% mobile phase B at a flow rate of 50 mL / minute and run in the below gradient programme.

[0091] Collect the fractions and check the purity limit NLT 85.0%. After complete elution of product wash the column with 90% Mobile Phase B [4 column volumes (2.0 L)].

[0092] Ammonium acetate purification process:

[0093] Column: UNISIL C18 (300*50 mm, lOp)

[0094] Sample preparation: Charge above pooled TFA run fractions into 5.0 L beaker, dilute with purified water.

[0095] Mobile phase A: Water (5 L) + Ammonium acetate salt (19.26 grams)

[0096] Mobile phase B: Acetonitrile (4.0 L) + Water (1.0 L)

[0097] Equilibrate the column with 35% mobile phase-B (2 column volumes 1.0 L) with a flow rate of 50mL / min.

[0098] Collect the fractions and check the purity limit NLT 98.0% and any single maximum impurity

[0099] NMT 0.50%.

[0100] Desalting Procedure

[0101] Column: UNISIL C18 (300*50 mm, 10g)

[0102] Sample preparation: Charge above pooled Ammonium acetate run fractions into 5.0 L beaker, dilute with purified water.

[0103] Mobile phase A: Water (5 L)

[0104] Mobile phase B: Acetonitrile (4.0 L) + water (1.0 L)

[0105] Equilibrate the column with 2% mobile phase-B (2 column volumes 1.0 L) with a flow rate of 50mL / min.

[0106] Collect the fractions and check the purity limit NLT 98.0% and any single maximum impurity NMT 0.50%.

[0107] While the present invention has been described in terms of its specific embodiments, certain modifications and equivalents will be apparent to those skilled in the art and are intended to be included within the scope of the present invention. The invention is illustrated below with reference to inventive and comparative examples and should not be construed to limit the scope of the invention. EXPERIMENTAL PORTION:

[0108] The details of the invention are given in the examples provided below, which are given to illustrate the invention only and therefore should not be construed to limit the scope of the invention.

[0109] Example 1: Process for the preparation of Tirzepatide by employing three fragments through hybrid approach.

[0110] Step-i: Synthesis of H-T(tBu)-F-T(tBu)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L- D(OtBu)-K(Boc)-I-A-Q(Trt)-K(Linker)-A-F-V-Q(Trt)-W(Boc)-L-I-A-G-G-P- S(tBu)-S(tBu)-G-A-P-P-P-S(tBu)-NH2compound of Formula IL

[0111] Fragment II of Fmoc-T(tBu)-F-T(tBu)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L- D(OtBu)-K(Boc)-I-A-Q(Trt)-K(Linker)-OH (3.0 grams) was dissolved in DMF (90 mL) then stirred for 10 minutes at 25-30 °C and coupled with Fragment III of H-A-F-V-Q(Trt)- W(Boc)-L-I-A-G-G-P-S(tBu)-S(tBu)-G-A-P-P-P-S(tBu)-NH2(2.4 grams) in the presence of EDC.HC1 (0.78 grams), HOBt (0.66 grams) and DMF at 25-30 °C and stirred for 4-7 hours at a temperature of 25-30 °C. The precipitated solid was extracted with ethyl acetate and washed with water and hexane. The resulting protected peptide was deprotected with tert-butylamine (1.72 mL), n-heptane in DMF (90 mL). Filtered the precipitated solid and washed with water and n-Heptane to get H-T(tBu)-F-T(tBu)- S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(Linker)-A-F- V-Q(Trt)-W(Boc)-L-I-A-G-G-P-S(tBu)-S(tBu)-G-A-P-P-P-S(tBu)-NH2compound of Formula II.

[0112] Step-ii: Synthesis of H-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(tBu)-S(tBu)-D(OtBu)- Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(Linker)-A-F-V-Q(Trt)- W(Boc)-L-I-A-G-G-P-S(tBu)-S(tBu)-G-A-P-P-P-S(tBu)-NH2compound of Formula III.

[0113] Fragment I of Fmoc-Y(tBu)-Aib-E(OtBu)-G-OH (0.43 grams) was dissolved in DMF (25.0 mL) then stirred for 10 minutes at 25-30 °C and coupled with H-T(tBu)-F-T(tBu)- S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(Linker)-A-F- V-Q(Trt)-W(Boc)-L-I-A-G-G-P-S(tBu)-S(tBu)-G-A-P-P-P-S(tBu)-NH2compound of Formula II (3.5 grams) in the presence of EDC.HC1 (0.58 grams), HOBt (0.49 grams) in DMF (27.5 mL) then stirred for 15-20 minutes at 5-10 °C, maintained for 4-7 hours at 25-30 °C. The precipitated solid was filtered and washed with water and n-heptane. The resulting protected peptide was deprotected with tert-butylamine (1.5 mL), n-heptane (50 mL) in DMF (52.5 mL). Filtered the precipitated solid and washed with water and DIPE to get H-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(tBu)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I- Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(Linker)-A-F-V-Q(Trt)-W(Boc)-L-I-A-G-G-P- S(tBu)-S(tBu)-G-A-P-P-P-S(tBu)-NH2 compound of Formula III.

[0114] Step-iii: Synthesis of crude Tirzepatide.

[0115] H-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(tBu)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L- D(OtBu)-K(Boc)-I-A-Q(Trt)-K(Linker)-A-F-V-Q(Trt)-W(Boc)-L-I-A-G-G-P-S(tBu)- S(tBu)-G-A-P-P-P-S(tBu)-NH2 compound of Formula III (3.0 grams) was cleaved with a cocktail mixture of TFA, TIPS, water and DTT (90% / 5% / 5% / 2.5%) 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 Tirzepatide.

[0116] Step-iv: Preparative HPLC purification of Tirzepatide.

[0117] Crude Tirzepatide (2.0 grams) was dissolved in 0.5 M ammonium formate loaded onto preparative C18 column (50x250 mm, 100 A0). The peptide was purified using a linear gradient of trifluoro acetic acid (0.1%) and acetonitrile: methanol (8: 1, 0.1% TFA) from 40% to 90% over 60 minutes. The pure fraction containing Tirzepatide was pooled. The acetonitrile was evaporated, and the aqueous layer was lyophilized to give the pure Tirzepatide as white solid. The resulting peptide was analysed by RP-HPLC and confirmed by MALDI or LC-MS.

Claims

We claim:

1. A process for the preparation of Tirzepatide compound of Formula I or a pharmaceutically acceptable salt thereof using a hybrid approach,Formula I which comprises: i) condensation of Fmoc-T(tBu)-F-T(tBu)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib- L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(Linker)-OH (Fragment II) with H-A-F-V- Q(Trt)-W(Boc)-L-I-A-G-G-P-S(tBu)-S(tBu)-G-A-P-P-P-S(tBu)-NH2(Fragment III) in the presence of coupling agent and solvent, followed by in-situ manner deprotection in the presence of base to obtain H-T(tBu)-F-T(tBu)-S(tBu)- D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(Linker)-A-F- V-Q(Trt)-W(Boc)-L-I-A-G-G-P-S(tBu)-S(tBu)-G-A-P-P-P-S(tBu)-NH2compound of Formula II; ii) condensing Fmoc-Y(tBu)-Aib-E(OtBu)-G-OH (Fragment I) with H-T(tBu)-F- T(tBu)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)- K(Linker)-A-F-V-Q(Trt)-W(Boc)-L-I-A-G-G-P-S(tBu)-S(tBu)-G-A-P-P-P- S(tBu)-NH2 compound of Formula II in the presence of coupling agent and solvent, followed by in-situ manner deprotection in the presence of base to obtain H-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(tBu)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I- Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(Linker)-A-F-V-Q(Trt)-W(Boc)-L-I-A-G- G-P-S(tBu)-S(tBu)-G-A-P-P-P-S(tBu)-NH2 compound of Formula III;iii) global deprotection of protected Tirzepatide compound of Formula III using a reagent and solvent to obtain crude Tirzepatide or a pharmaceutically acceptable salt thereof; iv) purifying the crude Tirzepatide by preparative HPLC to obtain pure Tirzepatide or a pharmaceutically acceptable salt thereof.

2. The process as claimed in claim 1, wherein said base is selected from the group consisting of 5-10% palladium on carbon, 2-10% hydrazine hydrate, triethylamine, diisopropyl ethyl amine, N-methyl morpholine, pyridine or a mixture thereof.

3. The process as claimed in claim 1, wherein said coupling agent is selected from the group consisting of Ethylcyano (hydroxyimino)acetate-2)-tri-(l-pyrrolidinyl)- Phosphonium hexa fluorophosphate (PyOxim), ethyl-2-cyano-2-(hydroxy amino) acetate (Oxyma pure), O-(benzotriazol-l-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), diisopropyl carbodiimide (DIC), 1,3- dicyclohexylcabodiimide (DCC), O-(7-azabenzotriazol- l-yl)-N,N,N',N'- tetramethyluronium hexafluorophosphate (HATU), 1 -(dimethyl aminopropyl)-3- ethylcarbodiimide hydrochloride (EDC HC1), O-(benzotriazol-l-yl)-l,l,3,3-tetra methyluronium hexafluorophosphate (HBTU), 1-Hydroxybenzo triazole (HOBt), Isopropyl chloro formate (IPCF), Benzotriazol- l-yl-oxy-tris(dimethyl-amino)- phosphonium hexa fluorophosphate (BOP), benzotriazole- 1- yloxytri(pyrrolidino)phosphonium hexa fluoro phosphate (PyBOP), N,N-bis-(2-oxo- 3-oxazolidinyl)phosphonic dichloride (BOP-CI), bromotri(pyrrolidino)phosphonium hexa fluoro phosphate (PyBrOP), O-(6-Chloro-l-hydrocibenzotriazol-l-yl)-l, 1,3,3- tetramethyl uranium tetra fluoroborate (TCTU), chlorotri (pyrrolidino)phosphonium hexafluorophosphate (PyClOP), Ethyl l,2-dihydro-2-ethoxyquinoline- carboxylate(EEDQ), isobutyl chloro formate (IBCF), 2-succinimido-l, 1,3,3- tetramethyluronium tetrafluoroborate(TSTU), l-Cyano-2-ethoxy-2-oxo ethylidene aminooxy) dimethyl amino morpholino-carbeniumhexafluorophosphate (COMU), 2- (5-norbomen-2,3-dicarboximido)- 1 , 1 ,3,3-tetramethyluronium tetrafluoroborate(TNTU), propane phosphonic acid anhydride (PPAA), 3-(diethoxy phosphoryloxy)- l,2,3-benzotriazin-4(3H)-one (DEPBT) or a mixture thereof.

4. The process as claimed in claim 1, wherein said solvent is selected from the groupconsisting of dimethylacetamide, dimethylformamide (DMF), formamide, N- Methylformamide, N-Methyl pyrrolidine (NMP), dimethyl acetamide (DM AC), methanol, ethanol, isopropanol, tert-Butanol, dichloromethane, dichloroethane, 1,4- dioxane, di-isopropyl ether, diethyl ether, tetrahydrofuran, methyl tert-butyl ether, ethyl-tert-butyl ether, ethyl acetate, isopropyl acetate, n-butyl acetate, isobutyl acetate, methyl acetate, acetonitrile, propionitrile, butyronitrile, isobutyronitrile, acetone, ethyl methyl ketone, methyl isobutyl ketone, diethyl ketone, pentane, n- hexane, n-heptane, water or a mixture thereof.

5. The process as claimed in claim 1, wherein the cleavage and global deprotection of the peptide is carried out with a cocktail mixture.

6. The process as claimed in claim 5, wherein the cocktail mixture consisting of TIPS, phenol, thioanisole, water or mixture thereof, preferably a mixture of TFA, TIPS, water and DTT.

7. A Fragment I: Fmoc-Y(tBu)-Aib-E(OtBu)-G-OH.Fmoc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-OHFragment I8. A Fragment II: Fmoc-T(tBu)-F-T(tBu)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L- D(OtBu)-K(Boc)-I-A-Q(Trt)-K(Linker)-OH.

9. A Fragment III: H-A-F-V-Q(Trt)-W(Boc)-L-I-A-G-G-P-S(tBu)-S(tBu)-G-A-P-P-P- S(tBu)-NH2.

10. A process for preparation of Tirzepatide compound of Formula I, comprising the use of individual fragments selected from the group consisting of:Fragment I: Fmoc-Y(tBu)-Aib-E(OtBu)-G-OH.Fragment II: Fmoc-T(tBu)-F-T(tBu)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(Linker)-OH.Fragment III: H-A-F-V-Q(Trt)-W(Boc)-L-I-A-G-G-P-S(tBu)-S(tBu)-G-A-P-P-P- S(tBu)-NH2.

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