WO2025052429A1

WO2025052429A1 - An improved process for the preparation of tirzepatide

Info

Publication number: WO2025052429A1
Application number: PCT/IN2024/051622
Authority: WO
Inventors: Mahender Rao Siripragada; Sunil Kumar Gandavadi; Shaik Shavali PARUMANCHALA; Hema Sundara Rao TELAKALA; Kalesha SHAIK
Original assignee: Neuland Laboratories Ltd
Current assignee: Neuland Laboratories Ltd
Priority date: 2023-09-04
Filing date: 2024-09-04
Publication date: 2025-03-13
Anticipated expiration: 2026-03-04

Abstract

An improved process for the preparation of Tirzepatide having the chemical structural Formula I. The present invention also relates to the compounds of SEQ ID NO. 1 to 14 or a pharmaceutically acceptable salt thereof for use in the synthesis of Tirzepatide or a pharmaceutically acceptable salt thereof. The present invention further relates to the intermediate compounds of Formulae II to Formulae XVIII and its process of preparation, which are used in the preparation of Tirzepatide or a pharmaceutically acceptable salt thereof.

Description

AN IMPROVED PROCESS FOR THE PREPARATION OF TIRZEPATIDE

Related Patent Application(s):

This application claims priority to and benefits from Indian provisional patent application No. 202341059209 filed on September 04, 2023; the disclosure of which is incorporated here by reference.

Title of the Invention

An improved process for the preparation of Tirzepatide or a pharmaceutically acceptable salt thereof.

Field of the Invention

The present invention relates to an improved process for the preparation of Tirzepatide or a pharmaceutically acceptable salt thereof, having the chemical structural Formula I.

Formula I

The present invention also relates to the novel compounds of SEQ ID NO. 1 to 14 or a pharmaceutically acceptable salt thereof for use in the synthesis of Tirzepatide or a pharmaceutically acceptable salt thereof.

The present invention further relates to the novel intermediate compounds of Formulae II to XVIII and its process of preparation, which are used in the preparation of Tirzepatide or a pharmaceutically acceptable salt thereof.

Background of the Invention

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 as show in three-letter code H-Tyr-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-Aib-Leu-Asp-Lys-Ile- Ala-Gln-Lys(AEEAc-AEEAc-y-Glu-Eicosanedioic acid)-Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala- Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NHz. The molecular formula is C225H348N48O68. 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.

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.

Tirzepatide is first disclosed in US 9474780 B2, this process leads to the formation of impurities and additional purification techniques required to get pure Tirzepatide. This process is highly expensive and commercially not viable.

Several process for preparation of Tirzepatide and its fragments have been disclosed in WO 2020/159949 Al, WO 2021/158444 Al, CN 112110981 A, CN 112661815 A, WO 2022079639 Al and WO 2021260530 Al.

In view of the above, there is a significant need to develop a cost-effective, stable, commercially viable, large scale and robust processes and intermediates to enable improved technology for production of highly pure Tirzepatide of Formula I with good yield.

Summary of the Invention

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

The present invention provides a cost effective, novel and an efficient process for the preparation of Tirzepatide or a pharmaceutically acceptable salt thereof and 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.

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,

Formula I which comprises: i) condensation of H-G-P-S(tBu)-S(tBu)-G-A-P-P-P-S(tBu)-NH₂ (SEQ ID NO. 1) with Fmoc-A-F-V-Q(Trt)-W(Boc)-L-I-A-G-OH (SEQ ID NO. 2) in presence of coupling agent and solvent, followed by in-situ manner deprotection in presence of base to obtain 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 compound of Formula II; ii) condensing 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 compound of Formula II with Fmoc-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)- Eicosanedioic acid mono-t-butyl)-OH (SEQ ID NO. 3) in presence of coupling agent and solvent, followed by in-situ manner deprotection in presence of base to obtain H-Q(Trt)- K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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) condensation of H-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono- t-butyl)-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 with Fmoc-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)- K(Boc)-I-A-OH (SEQ ID NO. 4) in presence of coupling agent and solvent, followed by in-situ manner deprotection in presence of base to obtain H-S(tBu)-D(OtBu)-Y(tBu)- S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)- Eicosanedioic acid mono-t-butyl)-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 IV; iv) condensation of H-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A- Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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 IV with Wl-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(W4)-OH (SEQ ID NO. 5) in presence of coupling agent and solvent, followed by in-situ manner deprotection in presence of base to obtain H-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(W4)-S(tBu)-D(OtBu)- Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a- OtBu)-Eicosanedioic acid mono-t-butyl)-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 (Protected Tirzepatide), where in W1 is Fmoc or CBZ and W4 is (tBu) or (Oxa); v) global deprotection of protected Tirzepatide using a reagent to obtain crude Tirzepatide or a pharmaceutically acceptable salt thereof; vi) purifying the crude Tirzepatide by preparative HPLC to obtain pure Tirzepatide or a pharmaceutically acceptable salt thereof.

In the second aspect, the present invention relates to a process for the preparation of Tirzepatide compound of Formula I or a pharmaceutically acceptable salt thereof,

Formula I which comprises: i) condensing H-G-P-S(tBu)-S(tBu)-G-A-P-P-P-S(tBu)-NH2 (SEQ ID NO. 1) with Fmoc- A-F-V-Q(Trt)-W(Boc)-L-I-A-G-OH (SEQ ID NO. 2) in presence of coupling agent and solvent, followed by in-situ manner deprotection in presence of base to obtain 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 compound of Formula II; ii) condensing 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 compound of Formula II with Fmoc-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)- Eicosanedioic acid mono-t-butyl)-OH (SEQ ID NO. 3) in presence of coupling agent and solvent, followed by in-situ manner deprotection in presence of base to obtain H-Q(Trt)- K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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) condensing H-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t- butyl)-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 with Fmoc-T(tBu)-F-T(tBu)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)- I-Aib-L-D(OtBu)-K(Boc)-I-A-OH (SEQ ID NO. 9) in presence of coupling agent and solvent, followed by in-situ manner deprotection in 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(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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 VI; iv) condensing 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(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t- butyl)-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 VI with W3-Y(tBu)-Aib-E(OtBu)-G-OH (SEQ ID NO. 10) in presence of coupling agent and solvent, followed by in-situ manner deprotection in 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(AEEAc-AEEAc-y-Glu (a- OtBu)-Eicosanedioic acid mono-t-butyl)-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 (Protected Tirzepatide), where in W3 is Fmoc, Boc or CBZ; v) global deprotection of protected Tirzepatide using a reagent to obtain crude Tirzepatide or a pharmaceutically acceptable salt thereof; vi) purifying the crude Tirzepatide by preparative HPLC to obtain pure Tirzepatide or a pharmaceutically acceptable salt thereof.

In the third aspect, the present invention relates to a process for the preparation of Tirzepatide compound of Formula I or a pharmaceutically acceptable salt thereof,

Formula I which comprises: i) condensing H-G-P-S(tBu)-S(tBu)-G-A-P-P-P-S(tBu)-NH2 (SEQ ID NO. 1) with Fmoc- Q(Trt)-K(W5)-A-F-V-Q(Trt)-W(Boc)-L-I-A-G-OH (SEQ ID NO. 6) in presence of coupling agent and solvent, followed by in-situ manner deprotection in presence of base to obtain H-Q(Trt)-K(W5)-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 VII, where in W5 is Dde or IVDde; ii) condensing H-Q(Trt)-K(W5)-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 VII with Fmoc-S(tBu)-D(OtBu)-Y(tBu)- S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-OH (SEQ ID NO. 7) in presence of coupling agent and solvent, followed by in-situ manner deprotection in presence of base to obtain H- S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(W5)-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 VIII, where in W5 is Dde or IVDde; iii) condensing H-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)- K(W5)-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 VIII with CBZ-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(W4)-OH (SEQ ID NO. 8) in presence of coupling agent and solvent to obtain Cbz-Y(tBu)-Aib- E(OtBu)-G-T(tBu)-F-T(W4)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)- K(Boc)-I-A-Q(Trt)-K(Dde or IVDde)-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 IX, where in W4 is (tBu) or (Oxa) and W5 is Dde or IVDde; iv) deprotection of the Dde or IVDe group in presence of base and then it was coupled with NH₂ to obtain Cbz-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(W4)-S(tBu)-D(OtBu)-Y(tBu)- S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(NH₂)-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 X; v) deprotection of amino group in presence of base and then it was coupled with Fmoc- AEEAc-AEEAc-OH in presence of coupling agent and solvent followed by in-situ manner Fmoc removal to obtain Cbz-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(W4)-S(tBu)- D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(AEEAc-AEEAc)-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 XI; vi) condensation of Cbz-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(W4)-S(tBu)-D(OtBu)-Y(tBu)- S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(AEEAc-AEEAc)-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 XI with Fmoc-Glu-OtBu in presence of coupling agent and solvent followed by in-situ manner Fmoc removal to obtain Cbz-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(W4)-S(tBu)- D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(AEEAc-AEEAc-y- Glu-OtBu)-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 XII; vii) condensation of Cbz-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(W4)-S(tBu)-D(OtBu)-Y(tBu)- S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(AEEAc-AEEAc-y-Glu-OtBu)-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 XII with eicosane dioic acid mono-t-butyl ester in presence of coupling agent and solvent to obtain Cbz-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(W4)-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 compound of Formula XIII; viii) deprotection of Cbz-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(W4)-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 compound of Formula XIII in presence of base to obtain H-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(W4)-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 (Protected Tirzepatide); ix) global deprotection of protected Tirzepatide using a reagent to obtain crude Tirzepatide or a pharmaceutically acceptable salt thereof; x) purifying the crude Tirzepatide by preparative HPLC to obtain pure Tirzepatide or a pharmaceutically acceptable salt thereof.

In the fourth aspect, the present invention relates to a process for the preparation of Tirzepatide compound of Formula I or a pharmaceutically acceptable salt thereof,

Formula I which comprises: i) condensation of H-G-P-S(tBu)-S(tBu)-G-A-P-P-P-S(tBu)-NH₂ (SEQ ID NO. 1) with Cbz-Q(Trt)-K(W5)-A-F-V-Q(Trt)-W(Boc)-L-I-A-G-OH (SEQ ID NO. 6) in presence of coupling agent and solvent, followed by in-situ manner deprotection in presence of base to obtain Cbz-Q(Trt)-K(W5)-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 XIV, where in W5 is Dde or IVDde; ii) deprotection of the Dde or IVDe group in presence of base and then it was coupled with NH₂ to obtain Cbz-Q(Trt)-K(NH₂)-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 XV; iii) deprotection of amino group in presence of base and then it was coupled with Fmoc- AEEAc-AEEAc-OH in presence of coupling agent and solvent, followed by in-situ manner Fmoc removal to obtain Cbz-Q(Trt)-K(AEEAc-AEEAc)-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 XVI; iv) condensation of Cbz-Q(Trt)-K(AEEAc-AEEAc)-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 XVI with Fmoc-Glu-OtBu in presence of coupling agent and solvent, followed by in-situ manner Fmoc removal to obtain Cbz-Q(Trt)-K(AEEAc-AEEAc-y-Glu-OtBu)-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 XVII; v) condensation of Cbz-Q(Trt)-K(AEEAc-AEEAc-y-Glu-OtBu)-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 XVII with eicosane dioic acid mono-t-butyl ester in presence of coupling agent and solvent, followed by in-situ manner Z group removal to obtain H-Q(Trt)-K(AEEAc-AEEAc-y- Glu-OtBu-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 compound of Formula XVIII; vi) condensation of H-Q(Trt)-K(AEEAc-AEEAc-y-Glu-OtBu-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 compound of Formula XVIII with Fmoc-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I- Aib-L-D(OtBu)-K(Boc)-I-A-OH (SEQ ID NO. 7) in presence of coupling agent and solvent, followed by in-situ manner deprotection in presence of base to obtain H-S(tBu)- D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(AEEAc-AEEAc-y- Glu-OtBu-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 compound of Formula XIX; vii) condensation of H-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A- Q(Trt)-K(AEEAc-AEEAc-y-Glu-OtBu-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 compound of Formula XIX with W3-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(W4)-OH (SEQ ID NO. 8) in presence of coupling agent and solvent, followed by in-situ manner deprotection in presence of base to obtain H-Y(tBu)-Aib-E(0tBu)-G-T(tBu)-F-T(W4)-S(tBu)-D(0tBu)-

Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(AEEAc-AEEAc-y-Glu-OtBu- 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 (Protected Tirzepatide), where in W4 is (tBu); viii) global deprotection of protected Tirzepatide using a reagent to obtain crude Tirzepatide or a pharmaceutically acceptable salt thereof; ix) purifying the crude Tirzepatide by preparative HPLC to obtain pure Tirzepatide or a pharmaceutically acceptable salt thereof.

In the fifth aspect, the present invention relates to a process for the preparation of Tirzepatide compound of Formula I or a pharmaceutically acceptable salt thereof,

Formula I which comprises: i) condensation of W2-Y(tBu)-Aib-E(0tBu)-G-T(tBu)-F-T(tBu)-S(tBu)-D(0tBu)-Y(tBu)- S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)- Eicosanedioic acid mono-t-butyl)-OH (SEQ ID NO. 11) 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 (SEQ ID NO. 12) in presence of coupling agent and solvent, followed by in-situ manner deprotection in presence of base to obtain (Protected Tirzepatide), where in W2 is Fmoc or Boc; ii) global deprotection of protected Tirzepatide using a reagent to obtain crude Tirzepatide or a pharmaceutically acceptable salt thereof; iii) purifying the crude Tirzepatide by preparative HPLC to obtain pure Tirzepatide or pharmaceutically acceptable salt thereof.

In the sixth aspect, the present invention relates to a process for the preparation of Tirzepatide compound of Formula I or a pharmaceutically acceptable salt thereof,

Formula I which comprises: i) condensation 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 (SEQ ID NO. 12) with Fmoc-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)- Eicosanedioic acid mono-t-butyl)-OH (SEQ ID NO. 3) in presence of coupling agent and solvent, followed by in-situ manner deprotection in presence of base to obtain H-Q(Trt)- K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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; ii) condensation of H-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono- t-butyl)-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 with W2-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-OH (SEQ ID NO. 13) in presence of coupling agent and solvent, followed by in-situ manner deprotection in 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(AEEAc-AEEAc-y-Glu (a- OtBu)-Eicosanedioic acid mono-t-butyl)-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 (Protected Tirzepatide), where in W2 is Fmoc or Boc; iii) global deprotection of protected Tirzepatide using a reagent 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 relates to the compound of SEQ ID NO. 1: H-G-P-S(tBu)-S(tBu)-G-A-P- P-P-S(tBu)-NH₂.

The present invention relates to the compound of SEQ ID NO. 2: Fmoc-A-F-V-Q(Trt)-W(Boc)- L-I-A-G-OH.

The present invention relates to the compound of SEQ ID NO. 3: Fmoc-Q(Trt)-K(AEEAc- AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-OH. The present invention relates to the compound of SEQ ID NO. 4: Fmoc-S(tBu)-D(OtBu)-

Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-OH.

The present invention relates to the compound of SEQ ID NO. 5: Wl-Y(tBu)-Aib-E(OtBu)-G- T(tBu)-F-T(W4)-OH, where in W1 is Fmoc or Cbz and W4 is (tBu) or (Oxa).

The present invention relates to the compound of SEQ ID NO. 6: Wl-Q(Trt)-K(W5)-A-F-V- Q(Trt)-W(Boc)-L-I-A-G-OH, where in W1 is Fmoc or Cbz and W5 is Dde or IVDde.

The present invention relates to the compound of SEQ ID NO. 7: Fmoc-S(tBu)-D(OtBu)-Y(tBu)- S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-OH.

The present invention relates to the compound of SEQ ID NO. 8: Cbz-Y(tBu)-Aib-E(OtBu)-G- T(tBu)-F-T(W4)-OH, where in W4 is (tBu) or (Oxa).

The present invention relates to the compound of SEQ ID NO. 9: Fmoc-T(tBu)-F-T(tBu)-S(tBu)- D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-OH.

The present invention relates to the compound of SEQ ID NO. 10: W3-Y(tBu)-Aib-E(OtBu)-G- OH, where in W3 is Fmoc, Boc or Cbz.

The present invention relates to the compound of SEQ ID NO. 11: W2-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 (a-OtBu)-Eicosanedioic acid mono-t-butyl)-OH, where in W2 is Fmoc or Boc.

The present invention relates to the compound of SEQ ID NO. 12: 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.

The present invention relates to the compound of SEQ ID NO. 13: W2-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-OH, where in W2 is Fmoc or Boc.

The present invention relates to the compound of SEQ ID NO. 14: Fmoc-Q(Trt)-K(AEEAc- AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-Ala-OH.

The present invention relates to the compound of Formula II: 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.

The present invention relates to the compound of Formula III: H-Q(Trt)-K(AEEAc-AEEAc-y- Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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.

The present invention relates to the compound of Formula IV: H-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)- I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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. The present invention relates to the compound of Formula V: H-Y(tBu)-Aib-E(OtBu)-G-T(tBu)- F-T(tBu/Oxa)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)- K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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 (Protected T irzepatide) .

The present invention relates to the compound of Formula VI: 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(AEEAc-AEEAc-y-Glu (a- OtBu)-Eicosanedioic acidmono-t-butyl)-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.

The present invention relates to the compound of Formula VII: H-Q(Trt)-K(W5)-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, where in W5 is Dde or IVDde.

The present invention relates to the compound of Formula VIII: H-S(tBu)-D(OtBu)-Y(tBu)- S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(W5)-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, where in W5 is Dde or IVDde.

The present invention relates to the compound of Formula IX: Cbz-Y(tBu)-Aib-E(OtBu)-G- T(tBu)-F-T(W4)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(W5)- 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, where in W4 is (tBu) or (Oxa) and W5 is Dde or IVDde.

The present invention relates to the compound of Formula X: Cbz-Y(tBu)-Aib-E(OtBu)-G- T(tBu)-F-T(W4)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)- K(NH2)-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, where in W4 is (tBu) or (Oxa).

The present invention relates to the compound of Formula XI: Cbz-Y(tBu)-Aib-E(OtBu)-G- T(tBu)-F-T(W4)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)- K(AEEAc-AEEAc)-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, where in W4 is (tBu) or (Oxa).

The present invention relates to the compound of Formula XII: Cbz-Y(tBu)-Aib-E(OtBu)-G- T(tBu)-F-T(W4)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)- K(AEEAc-AEEAc-y-Glu-OtBu)-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, where in W4 is (tBu) or (Oxa).

The present invention relates to the compound of Formula XIII: Cbz-Y(tBu)-Aib-E(OtBu)-G- T(tBu)-F-T(W4)-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, where in W4 is (tBu) or (Oxa). The present invention relates to the compound of Formula XIV: Cbz-Q(Trt)-K(W5)-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, where in W5 is Dde or IVDde.

The present invention relates to the compound of Formula XV: Cbz-Q(Trt)-K(NH2)-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.

The present invention relates to the compound of Formula XVI: Cbz-Q(Trt)-K(AEEAc-AEEAc)- 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.

The present invention relates to the compound of Formula XVII: Cbz-Q(Trt)-K(AEEAc-AEEAc- Y-Glu-OtBu)-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.

The present invention relates to the compound of Formula XVIII: H-Q(Trt)-K(AEEAc-AEEAc- Y-Glu-OtBu-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.

The present invention relates to the compound of Formula XIX: H-S(tBu)-D(OtBu)-Y(tBu)- S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(AEEAc-AEEAc-Y-Glu-OtBu-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.

The present invention relates to the compound of Formula XX: W3-Y(tBu)-Aib-E(OtBu)-G- T(tBu)-F-T(W4)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)- K(AEEAc-AEEAc-Y-Glu-OtBu-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, where in W3 is Fmoc, Boc or Cbz and W4 is (tBu) or (Oxa).

Brief Description of the Drawing(s)

Figure 1: Sequences as referred in the description.

Detailed Description of the Invention

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

The present invention relates to the compound of sequence ID’s, which are used in the preparation of Tirzepatide or a pharmaceutically acceptable salt are as follows:

SEQ ID NO. 1: H-G-P-S(tBu)-S(tBu)-G-A-P-P-P-S(tBu)-NH₂. SEQ ID NO. 2: Fmoc-A-F-V-Q(Trt)-W(Boc)-L-I-A-G-OH.

SEQ ID NO. 3 : Fmoc-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t- butyl)-OH.

SEQ ID NO. 4: Fmoc-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-OH.

SEQ ID NO. 5: Wl-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(tBu/Oxa)-OH, where in W1 is Fmoc or Cbz and W4 is (tBu) or (Oxa).

SEQ ID NO. 6: Wl-Q(Trt)-K(Dde or IVDde)-A-F-V-Q(Trt)-W(Boc)-L-I-A-G-OH, where in

W1 is Fmoc or Cbz.

SEQ ID NO. 7: Fmoc-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-OH.

SEQ ID NO. 8: Cbz-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(W4)-OH, where in W4 is (tBu) or (Oxa).

SEQ ID NO. 9: Fmoc-T(tBu)-F-T(tBu)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)- K(Boc)-I-A-OH.

SEQ ID NO. 10: W3-Y(tBu)-Aib-E(OtBu)-G-OH, where in W3 is Fmoc, Boc or Cbz.

SEQ ID NO. 11: W2-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 (a-OtBu)-Eicosanedioic acid mono-t-butyl)-OH, where in W2 is Fmoc or Boc.

SEQ ID NO. 12: 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.

SEQ ID NO. 13: W2-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-OH, where in W2 is Fmoc or Boc.

SEQ ID NO. 14: Fmoc-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t- butyl)-Ala-OH.

The present invention relates to the intermediate compounds, which are used in the preparation of Tirzepatide or a pharmaceutically acceptable salt are as follows:

Formula II: 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.

Formula III: H-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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.

Formula IV: H-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-

K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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.

Formula V: H-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(tBu/Oxa)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-

I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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 (Protected Tirzepatide).

Formula VI: 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(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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.

Formula VII: H-Q(Trt)-K(W5)-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, where in W5 is Dde or IVDde.

Formula VIII: H-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)- K(W5)-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, where in W5 is Dde or IVDde.

Formula IX: Cbz-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(W4)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-

Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(W5)-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, where in W4 is (tBu) or (Oxa) and W5 is Dde or IVDde.

Formula X: Cbz-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(W4)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-

Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(NH₂)-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, where in W4 is (tBu) or (Oxa).

Formula XI: Cbz-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(W4)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-

Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(AEEAc-AEEAc)-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, where in W4 is (tBu) or (Oxa).

Formula XII: Cbz-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(W4)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-

Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(AEEAc-AEEAc-y-Glu-OtBu)-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, where in W4 is (tBu) or (Oxa).

Formula XIII: Z-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(Oxa)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-

Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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, where in W4 is (tBu) or (Oxa).

Formula XIV: Cbz-Q(Trt)-K(W5)-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, where in W5 is Dde or IVDde.

Formula XV: Cbz-Q(Trt)-K(NH2)-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.

Formula XVI: Cbz-Q(Trt)-K(AEEAc-AEEAc)-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.

Formula XVII: Cbz-Q(Trt)-K(AEEAc-AEEAc-y-Glu-OtBu)-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. Formula XVIII: H-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t- butyl)-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.

Formula XIX: H-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-

Formula XX: W3-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(W4)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-

Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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, where in W3 is Fmoc, Boc or Cbz and W4 is (tBu) or (Oxa).

Peptide fragments are prepared by using solid phase peptide synthesis through linear approach.

Wherein SEQ ID abbreviation is defined as sequence identification.

The following three letter amino acid abbreviations are used throughout the invention, which are defined as follows:

Alanine: (Ala) A Arginine: (Arg) R

Asparagine: (Asn) N Aspartic acid: (Asp) D

Cysteine: (Cys) C Glutamine: (Gin) Q

Glutamic acid: (Glu) E Glycine: (Gly) G

Histidine: (His) H Isoleucine: (He) I

Leucine: (Leu) L Lysine: (Lys) K

Methionine: (Met) M Phenylalanine: (Phe) F

Proline: (Pro) P Serine: (Ser) S

Threonine: (Thr) T Tryptophan: (Tip) W

Tyrosine: (Tyr) Y Valine: (Vai) V

9-Fluorenylmethoxycarbonyl: Fmoc Di tert-butyl decarbonate: Boc

Trityl chloride: Trt Tert-butyl: tBu

Carboxybenzyl: Cbz Tert-butyl ester: OtBu

2-Aminoisobutyric acid: Aib Pseudoproline dipeptide Oxa

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

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, N- Dimethylformamide (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, N- Methylformamide, NMP, DMAC, methanol, ethanol, isopropanol, tert-Butanol, DCM, 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.

The coupling agent used throughout the reaction can 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), l-(dimethyl aminopropyl)-3-ethylcarbodiimide hydrochloride (EDC HC1), O-(benzotriazol-l-yl)-l,l,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,l,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- 1 , 1 ,3,3-tetramethyluronium tetrafluoroborate(TSTU), 1 -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 its mixture. The base used throughout the reaction can be selected from the group consisting of 5-10% palladium on carbon, 2-10% hydrazine hydrate, triethylamine, di-isopropyl ethyl amine, N- methyl morpholine or pyridine.

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.

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 using a cocktail mixture of TFA, TIPS, water and DTT (90%: 5%: 5%: 2.5%).

The protected amino acids are commercially available or may be prepared according to procedures known in the literature.

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.

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 well known in the art.

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 is as shown in Scheme-I. H-G-P-S(tBu)-S(tBu)-G-A-P-P-P-S(tBu)-NH₂ + Fmoc-A-F-V-Q(Trt)-W(Boc)-L-I-A-G-OH

H-A-F-V-Q(Trt)-W(Boc)-L-I-A-G-G-P-S(t

Formula

Fmoc-Q(Trt)-K(AEEAc-AEEAc-y-Glu(a-OtBu)- EDC.HC1, HOAt,

Eicosanedioic acid mono-t-butyl)-OH DMF

(SEQ ID NO. 3)

Fmoc-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-

H-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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 Formula III

Formula IV

Wl-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(W4)-OH

EDC.HC1, HOAt, (SEQ ID NO. 5) DMF where in W1 is Fmoc or Cbz & W4 is (tBu) or (Oxa)

'Fmoc/ Z-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(W4)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-' I-A-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-A-F-V-Q(Trt)-W(Boc)-L-I-

H-Y-Aib-E-G-T-F-T-S-D-Y-S-I-Aib-L-D-K-I-A-Q-K( AEEAc-AEEAc-y-Glu-Eicosanedioic acid)-A-F-

V-Q-W-L-I-A-G-G-P-S-S-G-A-P-P-P-S-NH₂ (Tirzepatide Pure) where in W1 is Fmoc or Cbz & W4 is (tBu) or (Oxa)

SCHEME-I In step i), condensation of H-G-P-S(tBu)-S(tBu)-G-A-P-P-P-S(tBu)-NH2 (SEQ ID NO. 1) with Fmoc-A-F-V-Q(Trt)-W(Boc)-L-I-A-G-OH (SEQ ID NO. 2) in presence of coupling agent and solvent followed by in-situ manner deprotection in presence of base to obtain 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 compound of Formula II.

The reaction temperature may range from 20 °C to 35 °C and 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.

In step ii), condensation 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 compound of Formula II with Fmoc-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)- Eicosanedioic acid mono-t-butyl)-OH (SEQ ID NO. 3) in presence of coupling agent and solvent followed by in-situ manner deprotection in presence of base to obtain H-Q(Trt)-K(AEEAc- AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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.

The reaction temperature may range from 20 °C to 35 °C and preferably at a temperature in the range from 25 °C to 30 °C. The duration of the reaction may range from 4 hour to 8 hours, preferably for a period of 7 hours.

In step iii), condensation of H-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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 with Fmoc-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)- I-A-OH (SEQ ID NO. 4) in presence of coupling agent and solvent followed by in-situ manner deprotection in presence of base to obtain H-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)- K(Boc)-I-A-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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 IV.

The reaction temperature may range from 20 °C to 35 °C and preferably at a temperature in the range from 25 °C to 30 °C. The duration of the reaction may range from 7 hour to 9 hours, preferably for a period of 8 hours. In step iv), condensation of H-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A- Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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 IV with Wl-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(W4)-OH (SEQ ID NO. 5) in presence of coupling agent and solvent followed by in-situ manner deprotection in presence of base to obtain H-Y(tBu)-Aib- E(OtBu)-G-T(tBu)-F-T(W4)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A- Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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 (Protected Tirzepatide).

The reaction temperature may range from 20 °C to 35 °C and 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.

In step v) global deprotection of protected Tirzepatide using a reagent to obtain crude Tirzepatide.

The reaction temperature may range from 20 °C to 35 °C and 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. The cleavage cocktail mixture consisting of TFA/TIPS/Water/DTT range from 70%/2.5%/2.5%/l% to 95%/10%/10%/5%, preferably cocktail mixture is 90%/5%/5%/2.5%.

In step vi) purifying the crude Tirzepatide by preparative HPLC to obtain pure Tirzepatide or a pharmaceutically acceptable salt thereof.

In another 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 is as shown in Scheme-II. H-G-P-S(tBu)-S(tBu)-G-A-P-P-P-S(tBu)-NH₂ + Fmoc-A-F-V-Q(Trt)-W(Boc)-L-I-A-G-OH

Fmoc-Q(Trt)-K(AEEAc-AEEAc-y-Glu(a-OtBu)-

Eicosanedioic acid mono-t-butyl)-OH EDC.HC1, HOAt,

(SEQ ID NO. 3) DMF

Fmoc-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-A-F-V-

Q(Trt)-W(Boc)-L-I-A-G-G-P-S(tBu)-S(tBu)-G-A-P-P-P-S(tBu)-NH

H-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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

Formula III

EDC.HC1, HOAt, Fmoc-T(tBu)-F-T(tBu)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)- DMF I-Aib-L-D(OtBu)-K(Boc)-I-A-OH (SEQ ID NO. 9)

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(AEEAc-

AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-A-F-V-Q(Trt)-W(Boc)-L-I-A-G-G-P-S(tBu)-

I-A-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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

Formula V

TFA, TIPS, DTT, Water

V-Q-W-L-I-A-G-G-P-S-S-G-A-P-P-P-S-NH₂ (Tirzepatide Crude)

Purification/ Lyophilization

V-Q-W-L-I-A-G-G-P-S-S-G-A-P-P-P-S-NH₂ (Tirzepatide Pure) where in W3 is Fmoc, Boc or Cbz

SCHEME-II In step i), condensation of H-G-P-S(tBu)-S(tBu)-G-A-P-P-P-S(tBu)-NH2 (SEQ ID NO. 1) with Fmoc-A-F-V-Q(Trt)-W(Boc)-L-I-A-G-OH (SEQ ID NO. 2) in presence of coupling agent and solvent followed by in-situ manner deprotection in presence of base to obtain 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 compound of Formula II.

The reaction temperature may range from 20 °C to 35 °C and preferably at a temperature in the range from 25°C to 30 °C. The duration of the reaction may range from 5 hours to 7 hours, preferably for a period of 6 hours.

The reaction temperature may range from 20 °C to 35 °C and preferably at a temperature in the range from 25 °C to 30 °C. The duration of the reaction may range from 2 hour to 4 hours, preferably for a period of 3 hours.

In step iii), condensation of H-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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 with Fmoc-T(tBu)-F-T(tBu)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L- D(OtBu)-K(Boc)-I-A-OH (SEQ ID NO. 9) in presence of coupling agent and solvent followed by in-situ manner deprotection in 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(AEEAc-AEEAc-y-Glu (a-OtBu)- Eicosanedioic acid mono-t-butyl)-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 VI.

The reaction temperature may range from 20 °C to 35 °C and preferably at a temperature in the range from 25 °C to 30 °C. The duration of the reaction may range from 6 hour to 8 hours, preferably for a period of 8 hours. In step iv), condensation 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(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t- butyl)-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 VI with W3-Y(tBu)-Aib-E(OtBu)-G-OH (SEQ ID NO. 10) in presence of coupling agent and solvent followed by in-situ manner deprotection in 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(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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 (Protected Tirzepatide).

In step v) global deprotection of protected Tirzepatide using a reagent to obtain crude Tirzepatide. The reaction temperature may range from 20 °C to 35 °C and 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. The cleavage cocktail mixture consisting of TFA/TIPS/Water/DTT range from 70%/2.5%/2.5%/l% to 95%/10%/10%/5%, preferably cocktail mixture is 90%/5%/5%/2.5%.

In another 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 is as shown in Scheme-Ill. H-G-P-S(tBu)-S(tBu)-G-A-P-P-P-S(tBu)-NH₂ + Fmoc-Q(Trt)-K(W5)-A-F-V-Q(Trt)- (SEQ ID NO. 1) W(Boc)-L-I-A-G-OH (SEQ ID NO. 6)

EDC HC1 HOAt where in W5 is Dde or IVDde DMF ,,

K(Boc)-I-A-Q(Trt)-K(AEEAc-AEEAc-y-Glu(a-OtBu)-Eicosanedioic acidmono-t-butyl)-A-F-V- Formula XIII

H-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(W4)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)- I-A-Q(Trt)-K(AEEAc-AEEAc-y-Glu(a-OtBu)-Eicosanedioic acid mono-t-butyl)-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

Formula V TIPS, DTT, Water

H-Y-Aib-E-G-T-F-T-S-D-Y-S-I-Aib-L-D-K-I-A-Q-K( AEEAc-AEEAc-y-Glu-Eicosanedioic acid)-A- F-V-Q-W-L-I-A-G-G-P-S-S-G-A-P-P-P-S-NH₂ (Tirzepatide Crude) Purification/ Lyophilization

H-Y-Aib-E-G-T-F-T-S-D-Y-S-I-Aib-L-D-K-I-A-Q-K( AEEAc-AEEAc-y-Glu-Eicosanedioic acid)-A-

F-V-Q-W-L-I-A-G-G-P-S-S-G-A-P-P-P-S-NH₂ (Tirzepatide Pure) where in W4 is (tBu) or (Oxa) and W5 is Dde or IVDde.

SCHEME-III

In step i), condensation of H-G-P-S(tBu)-S(tBu)-G-A-P-P-P-S(tBu)-NH₂ (SEQ ID NO. 1) with Fmoc-Q(Trt)-K(W5)-A-F-V-Q(Trt)-W(Boc)-L-I-A-G-OH (SEQ ID NO. 6) in presence of coupling agent and solvent, followed by in-situ manner deprotection in presence of base to obtain H-Q(Trt)-K(W5)-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 VII.

The reaction temperature may range from 20 °C to 35 °C and preferably at a temperature in the range from 25°C to 30 °C. The duration of the reaction may range from 3 hours to 5 hours, preferably for a period of 4 hours.

In step ii), condensation of H-Q(Trt)-K(W5)-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 VII with Fmoc-S(tBu)-D(OtBu)-Y(tBu)- S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-OH (SEQ ID NO. 7) in presence of coupling agent and solvent followed by in-situ manner deprotection in presence of base to obtain H-S(tBu)-D(OtBu)- Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(W5)-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 VIII.

The reaction temperature may range from 20 °C to 35 °C and preferably at a temperature in the range from 25 °C to 30 °C. The duration of the reaction may range from 4 hour to 8 hours, preferably for a period of 8 hours. In step iii), condensation of H-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A- Q(Trt)-K(W5)-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 VIII with Cbz-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(W4)-OH (SEQ ID NO. 8) in presence of coupling agent and solvent followed by in-situ manner deprotection in presence of base to obtain Cbz-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(W4)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)- I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(W5)-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 IX.

In step iv), deprotection of the Dde or IVDe group of Cbz-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F- T(W4)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(W5)-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 IX in presence of base followed by in-situ manner amination in presence of coupling agent and solvent to obtain Cbz-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(W4)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)- I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(NH₂)-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 X.

In step v), removal of the NH2 group of Cbz-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(W4)-S(tBu)- D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(W5)-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 X in presence of base followed by in-situ manner coupling of Fmoc-AEEAc-AEEAc in presence of coupling agent and solvent and removal of Fmoc in presence of base to obtain Cbz-Y(tBu)-Aib-E(OtBu)-G- T(tBu)-F-T(W4)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)- K(AEEAc-AEEAc)-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 XI. In step vi), condensation of Cbz-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(W4)-S(tBu)-D(OtBu)- Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(AEEAc-AEEAc)-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 XI with Fmoc-Glu-OtBu in presence of coupling agent and solvent to obtain Cbz-Y(tBu)-Aib-E(OtBu)- G-T(tBu)-F-T(W4)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)- K(AEEAc-AEEAc-y-Fmoc-Glu-OtBu)-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 XII.

In step vii), condensation of Cbz-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(W4)-S(tBu)-D(OtBu)- Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(AEEAc-AEEAc-y-Fmoc-Glu-OtBu)-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 XI with eicosane dioic acid mono-t-butyl ester in presence of coupling agent and solvent to obtain Cbz-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(W4)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L- D(OtBu)-K(Boc)-I-A-Q(Trt)-K(AEEAc-AEEAc-y-Glu- eicosane dioic acid 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 compound of Formula XIII.

In step viii), deprotection of Cbz-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(W4)-S(tBu)-D(OtBu)- Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)- Eicosanedioic acid mono-t-butyl)-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 XIII in presence of base to obtain H-Y(tBu)-Aib-E(OtBu)- G-T(tBu)-F-T(W4)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)- K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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 (Protected Tirzepatide).

In step ix) global deprotection of protected Tirzepatide using a reagent to obtain crude Tirzepatide or a pharmaceutically acceptable salt thereof;

In step x) purifying the crude Tirzepatide by preparative HPLC to obtain pure Tirzepatide or a pharmaceutically acceptable salt thereof.

In another 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 is as shown in Scheme-IV.

H-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)- 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 Formula XVIII

EDC.HC1, HOAt, Fmoc-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-

DMF D(OtBu)-K(Boc)-I-A-OH (SEQ ID NO. 7)

G-A-P-P-P-S(tBu)-NH₂ Formula XIX

K(Boc)-I-A-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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 [Protected Tirzepatide]

SCHEME-IV TFA, TIPS, DTT, Water

V-Q-W-L-I-A-G-G-P-S-S-G-A-P-P-P-S-NH₂ (Tirzepatide Crude) Lyophilization

H-Y-Aib-E-G-T-P-T-S-D-Y-S-I-Aib-L-D-K-I-A-Q-K(AEEAc-AEEAc-y-Glu-Eicosanedioic acid)-A-F- V-Q-W-L-I-A-G-G-P-S-S-G-A-P-P-P-S-NH₂ (Tirzepatide Pure)

SCHEME-IV

In step i), condensation of H-G-P-S(tBu)-S(tBu)-G-A-P-P-P-S(tBu)-NH2 (SEQ ID NO. 1) with Wl-Q(Trt)-K(W5)-A-F-V-Q(Trt)-W(Boc)-L-I-A-G-OH (SEQ ID NO. 6) in presence of coupling agent and solvent followed by in-situ manner deprotection in presence of base to obtain Wl- Q(Trt)-K(W5)-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 XIV, where in W1 is Fmoc or Cbz.

In step ii), deprotection of the Dde or IVDe group of Wl-Q(Trt)-K(W5)-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 XIV in presence of base followed by in-situ manner amination in presence of coupling agent and solvent to obtain Wl-Q(Trt)-K(NH₂)-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 XV.

In step iii), removal of the NH2 group of Wl-Q(Trt)-K(NH2)-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 XV in presence of base followed by in-situ manner coupling of Fmoc-AEEAc-AEEAc in presence of coupling agent and solvent and removal of Fmoc in presence of base to obtain Wl-Q(Trt)-K(AEEAc-AEEAc)-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 XVI. The reaction temperature may range from 20 °C to 35 °C and 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.

In step iv), condensation of Wl-Q(Trt)-K(AEEAc-AEEAc)-A-F-V-Q(Trt)-W(Boc)-E-I-A-G-G- P-S(tBu)-S(tBu)-G-A-P-P-P-S(tBu)-NH2 compound of Formula XVI with Fmoc-Glu-OtBu in presence of coupling agent and solvent to obtain Z-Q(Trt)-K(AEEAc-AEEAc-y-Fmoc-Glu- OtBu)-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 XVII.

In step v), condensation of Wl-Q(Trt)-K(AEEAc-AEEAc-y-Fmoc-Glu-OtBu)-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 XVII with eicosane dioic acid mono-t-butyl ester in presence of coupling agent and solvent to obtain H- Q(Trt)-K(AEEAc-AEEAc-Y-Glu- eicosane dioic acid 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 compound of Formula XVIII.

In step vi), condensation of H-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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 XVIII with Fmoc-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-E-D(OtBu)- K(Boc)-I-A-OH (SEQ ID NO. 7) in presence of coupling agent and solvent followed by in-situ manner deprotection in presence of base to obtain H-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-E- D(OtBu)-K(Boc)-I-A-Q(Trt)-K(AEEAc-AEEAc-Y-Glu (a-OtBu)-Eicosanedioic acid mono-t- butyl)-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 XIX.

In step vi), condensation of H-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-E-D(OtBu)-K(Boc)-I-A- Q(Trt)-K(AEEAc-AEEAc-Y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-A-F-V-Q(Trt)- W(Boc)-E-I-A-G-G-P-S(tBu)-S(tBu)-G-A-P-P-P-S(tBu)-NH2 compound of Formula XIX with Wl-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(tBu/Oxa)-OH (SEQ ID NO. 8) in presence of coupling agent and solvent followed by in-situ manner deprotection in presence of base to obtain H-Y(tBu)- Aib-E(OtBu)-G-T(tBu)-F-T(tBu/Oxa)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)- K(Boc)-I-A-Q(Trt)-K(AEEAc-AEEAc-Y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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 (Protected Tirzepatide). In step ix) global deprotection of protected Tirzepatide using a reagent to obtain crude Tirzepatide or a pharmaceutically acceptable salt thereof;

In another 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 is as shown in Scheme-V.

W2-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 (a-OtBu)-Eicosanedioic acid mono-t-butyl)-OH (SEQ ID NO. 11)

EDC HC1 HOAt H-A-F-V-Q(Trt)-W(Boc)-L-I-A-G-G-P-S(tBu)- DMF ’ ’ S(tBu)-G-A-P-P-P-S(tBu)-NH2 (SEQ ID NO. 12)

H-Y-Aib-E-G-T-F-T-S-D-Y-S-I-Aib-L-D-K-I-A-Q-Lys( AEEAc-AEEAc-y-Glu-Eicosanedioic acid)-A-F-

V-Q-W-L-I-A-G-G-P-S-S-G-A-P-P-P-S-NH₂ (Tirzepatide Pure) where in W2 is Fmoc or Boc

SCHEME-V

In step i), condensation of W2-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 (a-OtBu)- Eicosanedioic acid mono-t-butyl)-OH (SEQ ID NO. 11) 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 (SEQ ID NO. 12) in presence of coupling agent and solvent followed by in-situ manner deprotection in presence of base to obtain H-Y(tBu)-Aib- E(OtBu)-G-T(tBu)-F-T(Oxa)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A- Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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 (Protected Tirzepatide), where in W2 is Fmoc or Boc.

In step ii) global deprotection of protected Tirzepatide using a reagent to obtain crude Tirzepatide or a pharmaceutically acceptable salt thereof;

In step iii) purifying the crude Tirzepatide by preparative HPLC to obtain pure Tirzepatide or a pharmaceutically acceptable salt thereof. The reaction temperature may range from 20 °C to 35 °C and 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. The cleavage cocktail mixture consisting of TFA/TIPS/Water/DTT range from 70%/2.5%/2.5%/l% to 95%/10%/10%/5%, preferably cocktail mixture is 90%/5%/5%/2.5%.

In another 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 is as shown in Scheme-VI.

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

(SEQ ID NO. 12)

EDC.HC1, HOAt, Fmoc-Q(Trt)-K(Linker)-OH (SEQ ID NO. 3)

DMF

Q(Trt)-W(Boc)-L-I-A-G-G-P-S(tBu)-S(tBu)-G-A-P-P-P-S(tBu)-NH₂

I-A-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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 (Protected Tirzepatide)

TFA, TIPS, DTT, Water

H-Y-Aib-E-G-T-F-T-S-D-Y-S-I-Aib-L-D-K-I-A-Q-K( AEEAc-AEEAc-y-Glu-Eicosanedioic acid)-A-F- V-Q-W-L-I-A-G-G-P-S-S-G-A-P-P-P-S-NH₂ (Tirzepatide Crude) Lyophilization

H-Y-Aib-E-G-T-F-T-S-D-Y-S-I-Aib-L-D-K-I-A-Q-Lys( AEEAc-AEEAc-y-Glu-Eicosanedioic acid)-A- F-V-Q-W-L-I-A-G-G-P-S-S-G-A-P-P-P-S-NH₂ (Tirzepatide Pure)

SCHEME-VI

In step i), condensation 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 (SEQ ID NO. 12) with Fmoc-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)-

Eicosanedioic acid mono-t-butyl)-OH (SEQ ID NO. 03) in presence of coupling agent and solvent followed by in-situ manner deprotection in presence of base to obtain H-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 compound of Formula III.

In step ii), condensation of H-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 compound of Formula III with W2-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-OH (SEQ ID NO. 13) in presence of coupling agent and solvent followed by in-situ manner deprotection in 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(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 (Protected Tirzepatide), where in W2 is Fmoc or Boc;

In step iii) purifying the crude Tirzepatide by preparative HPLC to obtain pure Tirzepatide or a pharmaceutically acceptable salt thereof.

Preparative HPLC method for purification of Tirzepatide:

Trifluoroacetic acid purification:

Sample preparation: 5 Grams of crude Tirzepatide was dissolved in 800 mL of water and 25 % aqueous ammonia solution added dropwise to get the clear solution.

Column: YMC Triart (50x250 mm, 10 pm)

Mobile phase- A: Tri fluoro acetic acid (5 mL) + water (5 mL).

Mobile phase-B: Isopropyl alcohol (2.5 mL) + Acetonitrile (2.5 mL) + Ortho phosphoric acid (5 mL)

Equilibrate the column with 5% mobile phase B at a flow rate of 60 mL/minute.

Ammonium bicarbonate purification process:

Fraction obtained from the above purification process is diluted with water.

Mobile phase-A: water (5 Ltr) + Ammonium bicarbonate (8.0 gms); Mobile phase-B: Acetonitrile: water (8:2)

Equilibrate the column with 5 % mobile phase-B with a flow rate of 50mL/min.

Collect the fractions as 25mL/vial and pooled fraction was lyophilized to get the pure Tirzepatide.

Purity: 97.2 %

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:

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.

Example 1: Process for the preparation of Tirzepatide by employing four fragments through hybrid approach.

Step-i: Synthesis 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)-NHi compound of Formula II.

Fmoc-A-f-V-Q(Trt)-W(Boc)-L-I-A-G-OH (14.7 grams) (SEQ ID: 2) was dissolved in DMF (180 mL) and stirred for 10 minutes at 25-30 °C. H-G-P-S(tBu)-S(tBu)-G-A-P-P-P-S(tBu)-NH2 (13.0 grams) (SEQ ID: 1), EDC.HC1 (4.85 grams) and HOAT (4.3 grams) in DMF was added to the resulting reaction mixture at 5-10 °C and stirred for 3-5 hours at the 25-30 °C temperature. Precipitated solid was extracted with ethyl acetate and washed with water. The resulting protected peptide was deprotected with tert-butylamine (10.0 mL), n-heptane in DMF. Filtered the precipitated solid and washed with water and hexane to get 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 compound of Formula II. Step-ii: Synthesis of H-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t- butyl)-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.

Fmoc-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-OH (10.0 grams) (SEQ ID: 3) was dissolved in DMF (112 mL) then coupled 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 compound of Formula II in presence of EDC.HC1 (3.65 grams), HOAt (3.5 grams) in DMF (180.0 mL) and stirred for 10 minutes at 5-10 °C temperature, maintain for 4-7 hours at 25-30 °C temperature to obtain protected peptide. The resulting protected peptide was deprotected with tert-butylamine (4.8 mL), n-heptane in DMF (70.0 mL) maintained for 2-3 hours at 25-30 °C. Filtered the precipitated solid and washed with water and DIPE to get H-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t- butyl)-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.

Step-iii: Synthesis of H-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A- Q(Trt)-K(AEEAc-AEEAc-Y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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 IV.

Fmoc-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-OH (3.1 grams) (SEQ ID: 4) was dissolved in DMF (150 mL) then coupled with H-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a- OtBu)-Eicosanedioic acid mono-t-butyl)-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 in presence of EDC.HC1 (1.35 grams), HOAt (1.1 grams) and stirred for 10-15 minutes at 5-10 °C, maintain for 12 hours at 25-30 °C temperature. Precipitated solid was filtered and washed with water and hexane. The resulting protected peptide was deprotected with tert-butylamine (2.2 mL), n-heptane (40 mL) in DMF. Filtered the precipitated solid and washed with water, hexane and DIPE to get H-S(tBu)-D(OtBu)-Y(tBu)- S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(AEEAc-AEEAc-Y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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 IV.

Step-iv: Synthesis of H-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(W4)-S(tBu)-D(OtBu)-Y(tBu)- S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(AEEAc-AEEAc-Y-Glu (a-OtBu)-

Eicosanedioic acid mono-t-butyl)-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 V.

Wl-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(W4)-OH (8 grams) (SEQ ID: 5) was dissolved in DMF (100 mL) then coupled with H-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A- Q(Trt)-K(AEEAc-AEEAc-Y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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 IV (5.0 grams) in presence of EDC.HC1 (0.5 grams), HOAt (0.4 grams) in DMF, stirred for 15-20 minutes at 5-10 °C, maintain for 4-7 hours at 25-30 °C. Precipitated solid was filtered and washed with water and hexane. The resulting protected peptide was deprotected with tert-butylamine (1.5 mL), n-heptane (50 mL) in DMF. Filtered the precipitated solid and washed with water, hexane and methanol to get H-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(W4)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I- Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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 V.

Step-v: Synthesis of crude Tirzepatide.

H-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(W4)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)- K(Boc)-I-A-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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 V (4.0 grams) was cleaved with a cocktail mixture of TFA, TIPS, water and DTT (90%/5%/5%/2.5%) 10-15 °C and stirred for 3-6 hours at the same temperature. Chilled MTBE 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.

Step-vi: Synthesis of pure Tirzepatide.

Crude Tirzepatide (10.0 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 the 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.

Yield: 1.0 gram.

Example 2: Process for the preparation of Tirzepatide by employing five fragments through hybrid approach.

Step-i: Synthesis 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 compound of Formula II.

Fmoc-A-F-V-Q(Trt)-W(Boc)-L-I-A-G-OH (19.5 grams) (SEQ ID: 2) was dissolved in DMF (180 mL) and stirred for 10 minutes at 25-30 °C. H-G-P-S(tBu)-S(tBu)-G-A-P-P-P-S(tBu)-NH2 (13 grams) (SEQ ID: 1), EDC.HC1 (5.4 grams) and HOAT (4.3 grams) added to the reaction mixture at 5-10 °C and stirred for 3-5 hours at the 25-30 °C. The resulted protected peptide was subjected to treatment with tert-butylamine (10 mL), n-heptane (70 mL). Filtered the precipitated solid and washed with water and hexane to get 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 compound of Formula II.

Step-ii: Synthesis of H-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t- butyl)-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.

Fmoc-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-OH (3.3 grams) (SEQ ID: 3) was dissolved in DMF (75 mL) then coupled 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 compound of Formula II (5.0 grams) in presence of EDC.HC1 (1.2 grams), HOAt (1.1 grams) stirred for 10 minutes at 5-10 °C, maintain for 4-7 hours at 25-30 °C to obtain protected peptide. The resulted protected peptide was subjected to treatment with tert-butylamine (1.0 mL), n-heptane (35.0 mL) maintained for 2-3 hours at 25- 30 °C. Filtered the precipitated solid and washed with water and DIPE to get H-Q(Trt)- K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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.

Step-iii: 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(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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 VI.

Fmoc-T(tBu)-F-T(tBu)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-OH (3.0 grams) (SEQ ID: 9) was dissolved in DMF (75 mL) then coupled with H-Q(Trt)-K(AEEAc- AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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 (5.0 grams) in presence of EDC.HC1 (1.0 grams), HOAt (0.8 grams) stirred for 10-15 minutes at 5-10 °C, maintain for 25- 30 hours at 25-30 °C. Precipitated solid was filtered and washed with water and hexane. The resulted protected peptide was subjected to treatment with tert-butylamine (0.8 mL), n-heptane (35 mL), Filtered the precipitated solid and washed with water, hexane and DIPE 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(AEEAc- AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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 VI.

Step-iv: 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(AEEAc-AEEAc-y-Glu (a-OtBu)-

Eicosanedioic acid mono-t-butyl)-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 V. W3-Y(tBu)-Aib-E(0tBu)-G-0H (2.1 grams) (SEQ ID: 10) was dissolved in DMF (300 mL) then 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(AEEAc-AEEAc-Y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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 VI (15.0 grams) in presence of EDC.HC1 (1.5 grams), HOAt (1.36 grams) stirred for 15-20 minutes at 5- 10 °C, maintain for 4-7 hours at 25-30 °C. Precipitated solid was filtered and washed with water and hexane. The resulted protected peptide was subjected to treatment with tert -butylamine (1.6 mL), n-heptane (105 mF). Filtered the precipitated solid and washed with water, hexane and methanol 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(AEEAc-AEEAc-Y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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 V.

Step-v: Synthesis of crude Tirzepatide.

Protected peptide (4.0 grams) was subjected to global cleavage in the presence of cocktail mixture (TFA, TIPS, water and DTT in the ratio of 87.5:5:5:2.5) at 10 tol5 °C for 2 hours under stirring. To this chilled MTBE was added and stirred for 2 hours, the precipitated solid was filtered and washed with DCM followed by DIPE to obtain crude Tirzepatide as solid.

Step-vi: Synthesis of pure Tirzepatide.

Crude Tirzepatide (10 grams) was dissolved in 0.5 M ammonium formate and loaded onto preparative C18 column (50x250 mm, 100 A0). The peptide was purified using a linear gradient of trifluoroacetic acid (0.1 %) and acetonitrile: methanol (8:1, 0.1% TFA) from 40 % to 90 % over 60 minutes. The pure fraction containing the Tirzepatide was pooled. The acetonitrile was evaporated, and the aqueous layer was lyophilized to give the Tirzepatide as white solid. The resulting peptide was analysed by RP-HPLC and confirmed by MALDI or LC-MS.

Yield: 1.0 gram.

Example 3: Process for the preparation of Tirzepatide by employing Four Fragments through hybrid approach.

Step-i: Synthesis of H-Q(Trt)-K(W5)-A-F-V-Q(Trt)-W(Boc)-L-I-A-G-G-P-S(tBu)-S(tBu)- G-A-P-P-P-Ser(tBu)-NH2 compound of Formula VII.

Fmoc-Q(Trt)-K(W5)-A-P-V-(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-OH (21.0 grams) (SEQ ID: 6) was dissolved in DMF (112 mL) then coupled with H-G-P-S(tBu)-S(tBu)-G-A-P-P-P-S(tBu)-NH2 (10.0 grams) (SEQ ID: 1) in presence of EDC.HC1 (7.5 grams), HOAt (6.1 grams) and stirred for 10 minutes at 5-10 °C , maintain for 4-7 hours at 25-30 °C temperature to obtain protected peptide. The resulting protected peptide was subjected to treatment with tert -butylamine (6.2 mL), n- heptane in DMF (50.0 mL) maintained for 2-3 hours at 25-30°C. Filtered the precipitated solid and washed with water and DIPE to get H-Q(Trt)-K(W5)-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 VII.

Step-ii) Synthesis of H-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A- Q(Trt)-K(W5)-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 VIII.

Fmoc-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-OH (12.4 grams) (SEQ ID: 7) was dissolved in DMF (112 mL) then coupled with H-Q(Trt)-K(W5)-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 VII (20.0 grams) in presence of EDC.HC1 (5.2 grams), HOAt (4.6 grams) and stirred for 10 minutes at 5-10 °C, maintain for 4-7 hours at 25-30 °C to obtain protected peptide. The resulted protected peptide was subjected to treatment with tert-butylamine (4.3 mL), n-heptane in DMF (100.0 mL) maintained for 2-3 hours at 25-30 °C. Filtered the precipitated solid and washed with water and DIPE to get H-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(W5)-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 VIII.

Step-iii) Synthesis of Cbz-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(W5)-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 IX.

Cbz-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(tBu)-OH (4.9 grams) (SEQ ID: 8) was dissolved in DMF (375 mL) then coupled with H-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K (Boc)- I-A- Q(Trt)-K(Dde or IVDde)-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 VIII (20.0 grams) in presence of EDC.HC1 (3.4 grams), HOAt (3.0 grams) and stirred for 10-15 minutes at 5-10 °C, maintain for 25-30 hours at 25-30 °C. Reaction mass precipitate with water. Filtered the precipitated solid and washed with water, hexane and DIPE to get Cbz-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(W5)-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 IX.

Step-iv: Synthesis of Cbz-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 (Dde or IVDde)-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 X.

Cbz-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(W5)-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 IX. (20.0 grams) was dissolved in DMF (300 mL) stirred for 15-20 minutes at 5-10 °C. Slowly add 2 % Hydrazine hydrate solution (20 mL) maintain for 4-7 hours at 25-30 °C. Precipitated solid was filtered and washed with water and hexane. The resulted protected peptide was filtered the precipitated solid and washed with water, hexane and methanol to get Z-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 (NH2)-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 X.

Step-v: Synthesis of Cbz-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)-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 XI.

Cbz-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(NH₂)-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 X (15.0 grams) was dissolved in DMF (200 mL) then coupled with Fmoc-AEEAc-AEEAc-OH (1.5 grams) in presence of EDC.HC1 (2.2 grams), HOAt (2.0 grams) and stirred for 15-20 minutes at 5-10 °C, maintain for 4-7 hours at 25-30 °C. The resulted protected peptide was subjected to treatment with tert-butylamine (7.5 mL), n-heptane (105 mL). Filtered the precipitated solid and washed with water, hexane and methanol to get Cbz- 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)-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 XI.

Step-vi: Synthesis of Cbz-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-E-OtBu)-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 XII.

Cbz-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)-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 XI (12.0 grams) was dissolved in DMF (200 mL) charge 0.9 grams of Fmoc-Glu-OtBu into the above solution, then coupled in presence of EDC.HC1 (1.6 grams), HOAt (1.3 grams) and stirred for 15-20 minutes at 5-10 °C, maintain for 4-7 hours at 25-30 °C. The resulted protected peptide was subjected to treatment with tert- butylamine (1.2 mL), n-heptane (60 mL). Filtered the precipitated solid and washed with water, hexane and methanol to get Z-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-E-OtBu)-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 XII. Step-vii: Synthesis of Cbz-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-E-OtBu- Eicosane dioic acid 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)-NHz compound of Formula XIII.

Cbz-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-E-OtBu)-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 XII (10.0 grams) was dissolved in DMF (150 mL) charge 0.8 grams of Eicosane dioic acid mono-t-Butyl ester into the above solution, then coupled in presence of EDC.HC1 (1.3 grams), HOAt (1.1 grams) and stirred for 15- 20 minutes at 5-10 °C, maintain for 4-7 hours at 25-30 °C to obtain Cbz-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-E-OtBu-Eicosane dioic acid 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 compound of Formula XIII.

Step-viii: 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(AEEAc-AEEAc-E-OtBu- Eicosane dioic acid 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 compound of Formula V.

Cbz-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-E-OtBu)-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 VIII (9.5 grams) dissolved in Methanol /DMF (190 mL), under nitrogen atmosphere charge 5% palladium on carbon (2.2 grams). The resulted peptide was isolated with water. Filtered the precipitated solid and washed with water, hexane and methanol 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(AEEAc-AEEAc-E-OtBu- Eicosane dioic acid 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 compound of Formula V.

Step-ix: Synthesis of crude Tirzepatide.

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(AEEAc-AEEAc-E-OtBu-Eicosane dioic acid 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 compound of Formula V (4.0 grams) was subjected to global cleavage in the presence of cocktail mixture (TFA, TIPS, water and DTT in the ratio of 87.5:5:5:2.5) at 10 tol5 °C for 2 hours under stirring. To this chilled MTBE was added and stirred for 2 hours, the precipitated solid was filtered and washed with DCM followed by DIPE to obtain crude Tirzepatide as solid. Step-x: Synthesis of pure Tirzepatide.

Crude Tirzepatide (10 grams) was dissolved in 0.5 M ammonium formate and loaded onto preparative Cl 8 column (50x250 mm, 100 A⁰). The peptide was purified using a linear gradient of trifluoroacetic acid (0.1 %) and acetonitrile: methanol (8:1, 0.1% TFA) from 40 % to 90 % over 60 minutes. The pure fraction containing the Tirzepatide was pooled. The acetonitrile was evaporated, and the aqueous layer was lyophilized to give the Tirzepatide as white solid. The resulting peptide was analysed by RP-HPLC and confirmed by MALDI or LC-MS.

Yield: 1.0 gram.

Example 4: Process for the preparation of Tirzepatide by using four fragments through hybrid approach.

Step-i: Synthesis of Wl-Q(Trt)-K(W5)-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 XIV.

Wl-Q(Trt)-K(W5)-A-F-V-Q (Trt)-W(Boc)-L-I-A-G-OH (9.3 grams) (SEQ ID: 6) was dissolved in DMF (150 mL) and stirred for 10 minutes at 25-30 °C. H-G-P-S(tBu)-S(tBu)-G-A-P-P-P- S(tBu)-NH2 (10.0 grams) (SEQ ID: 1), EDC.HC1 (2.5 grams) and HOAT (2.3 grams) in reaction mixture at 5-10 °C and raised to 25-30 °C stirred for 3-5 hours. Reaction mass precipitated with water. Filtered the precipitated solid and washed with water and hexane to get Wl-Q(Trt)- K(W5)-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 XIV.

Step-ii: Synthesis of Wl-Q(Trt)-K(NH₂)-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 XV.

Wl-Q(Trt)-K(W5)-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 XIV (10.0 grams) dissolved in (80 mL) of DMF. Cool the contents to 5-10 °C. stirred for 10 minutes at 5-10 °C, slowly add 2 % Hydrazine hydrate solution (25 mL) to reaction mass. Maintain for 4-7 hours at 25-30 °C. The resulted protected peptide was precipitated with water, stir for 2-3 hours at 25-30 °C. Filtered the precipitated solid and washed with water and DIPE to get Wl-Q(Trt)-K (NH₂)-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 XV.

Step-iii: Synthesis of Wl-Q(Trt)-K(AEEAc-AEEAc)-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 XVI.

Wl-Q(Trt)-K(NH₂)-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 XV (7.0 grams) was dissolved in DMF (85 mL) then coupled with Fmoc-AEEAc-AEEAc-OH (0.86 grams) in presence of EDC.HC1 (0.92 g), HOAt (0.87 g) stirred for 10-15 minutes at 5-10 °C, maintain for 25-30 hours at 25-30 °C. The resulted protected peptide was subjected to treatment with tert-butylamine (1 mL), n-heptane (35 mL). Filtered the precipitated solid and washed with water, hexane and DIPE to get Wl-Q(Trt)-K(AEEAc- AEEAc)-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 XVI.

Step-iv: Synthesis of Wl-Q(Trt)-K(AEEAc-AEEAc-Glu-OtBu)-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 XVII.

Wl-Q(Trt)-K(AEEAc-AEEAc)-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 XVI (6.0 grams) was dissolved in DMF (525 mL) then coupled with Fmoc-Glu-OtBu (0.55 grams) in presence of EDC.HC1 (1.7 grams), HOAt (1.4 grams) and stirred for 15-20 minutes at 5-10 °C, maintain for 4-7 h at 25-30 °C. The resulted protected peptide was subjected to treatment with tert-butylamine (0.7 mL), n-heptane (30 mL). Filtered the precipitated solid and washed with water, hexane and methanol to get Wl-Q(Trt)- K(AEEAc-AEEAc-E-OtBu)-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-XVII.

Step-v: Synthesis of H-Q(Trt)-K(AEEAc-AEEAc-E-OtBu-Eicosane dioic acid 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 compound of Formula VIII.

Wl-Q(Trt)-K(AEEAc-AEEAc-E-OtBu)-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-XVII (5.0 grams) was dissolved in DMF (60 mL) then coupled with Eicosane dioic acid mono-t-Butyl ester (0.5 grams) in presence of EDC.HC1 (0.8 grams), HOAt (0.7 grams) in DMF and stirred for 15-20 minutes at 5-10 °C, maintain for 4- 7 hours at 25-30 °C. Reaction mass precipitated with water and bed wash with MTBE. Filtered the precipitated solid and washed with water, hexane and methanol to get H-Q(Trt)-K(AEEAc- AEEAc-E-OtBu-Eicosane dioic acid 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 compound of Formula XVIII.

Step-vi: Synthesis of H-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A- Q(Trt)-K(AEEAc-AEEAc-Glu-OtBu-Eicosane dioic acid 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 compound of Formula XIX.

Fmoc-S(tBu)-D(OtBu-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-OH (2.5 grams) (SEQ ID: 7) was dissolved in DMF (105 mL) then coupled with H-Q(Trt)-K(AEEAc-AEEAc-Glu-OtBu- Eicosane dioic acid 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 compound of Formula-XVIII (7.0 grams) in presence of EDC.HC1 (1.1 grams), HOAt (0.9 grams) in DMF and stirred for 15-20 minutes at 5-10 °C, maintain for 4-7 hours at 25-30 °C. The resulted protected peptide was subjected to treatment with tert-butylamine (1.0 mL), n-heptane (40 mL). Filtered the precipitated solid and washed with water, hexane and methanol to get H-S(tBu)-D(OtBu-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)- K(AEEAc-AEEAc-Glu-OtBu-Eicosane dioic acid 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 compound of Formula-XIX.

Step-vii: Synthesis of H-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(W4)-S(tBu)-D(OtBu)-Y(tBu)- S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(AEEAc-AEEAc-Glu-OtBu-Eicosane dioic acid 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 compound of Formula V.

Fmoc-Y(tBu)-Aib-E(OtBu-G-T(tBu)-F-T(W4)-OH (1.3 grams) (SEQ ID-8) was dissolved in DMF (525 mL) then coupled with H-S(tBu)-D(OtBu-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)- I-A-Q(Trt)-K(AEEAc-AEEAc-Glu-OtBu-Eicosane dioic acid 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 compound of Formula- XIX (7.0 grams) in presence of EDC.HC1 (0.8 grams), HOAt (0.7 grams) and stirred for 15-20 minutes at 5-10 °C, maintain for 4-7 hours at 25-30 °C. The resulted protected peptide was subjected to treatment with tert-butylamine (0.7 mL), n-heptane (35 mL). Filtered the precipitated solid and washed with water, hexane and methanol to get H-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F- T(W4)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(AEEAc- AEEAc-Glu-OtBu-Eicosane dioic acid 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 compound of Formula V.

Step-viii: Synthesis of crude Tirzepatide.

Protected peptide (4.o grams) was subjected to global cleavage in the presence of cocktail mixture (TFA, TIPS, water and DTT in the ratio of 87.5:5:5:2.5) at 10 tol5 °C for 2 hours under stirring. To this chilled MTBE was added and stirred for 2 hours, the precipitated solid was filtered and washed with DCM followed by DIPE to obtain crude Tirzepatide as solid.

Step-ix: Synthesis of pure Tirzepatide.

Crude Tirzepatide 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 the Tirzepatide was pooled. The acetonitrile was evaporated and the aqueous layer was lyophilized to give the Tirzepatide as white solid. The resulting peptide was analysed by RP-HPLC and confirmed by MALDI or LC-MS.

Yield: 1.0 gram. Example 5: Process for the preparation of Tirzepatide by employing block approach through hybrid approach.

Step-i: 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(AEEAc-AEEAc-y-Glu (a-OtBu)-

W2-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 (a-OtBu)-Eicosanedioic acid mono-t- butyl)-OH (25 grams) (SEQ ID: 11) was dissolved in DMF (300 mL) coupled 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 (18.0 grams) (SEQ ID: 12) in presence of EDC.HC1 (3.7 grams), HOAt (3.1 grams), stirred for 15-20 minutes at 5-10 °C, maintain for 4-7 hours at 25-30 °C. The resulted protected peptide was subjected to treatment with tert-butylamine (4.0 mL), n-heptane (100 mL). Filtered the precipitated solid and washed with water, hexane and methanol 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(AEEAc-AEEAc-y-Glu (a- OtBu)-Eicosanedioic acid mono-t-butyl)-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 V.

Step-ii: Synthesis of crude Tirzepatide

Step-iii: Synthesis of pure Tirzepatide.

Yield: 1.0 grams.

Example 6: Process for the preparation of Tirzepatide by employing block approach through hybrid approach. Step-i: Synthesis of H-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t- butyl)-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.

Fmoc-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-OH (10.0 grams) (SEQ ID: 3) was dissolved in DMF (350 mL) and stirred for 10 minutes at 25-30 °C. 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 (18.0 g) (SEQ ID: 12), EDC.HC1 (4.4 grams) and HOAT (4.1 grams) in DMF was added to the reaction mixture at 5-10 °C and stirred for 3-5 hours at the 25-30 °C. The resulted protected peptide was subjected to treatment with tert-butylamine (4.3 mL), n-heptane. Filtered the precipitated solid and washed with water and hexane to get H-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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.

Step-ii: Synthesis of H-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(tBu/Oxa)-S(tBu)-D (OtBu)- Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)- Eicosanedioic acid mono-t-butyl)-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 V.

W2-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(tBu/Oxa)-S(tBu)-D (OtBu)-Y(tBu)-S(tBu)-I-Aib-L- D(OtBu)-K(Boc)-I-A-OH (3.0 grams) (SEQ ID: 13) was dissolved in DMF (50 mL) then coupled with H-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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) in presence of EDC.HC1 (0.6 grams), HOAt (0.5 grams) and stirred for 10-15 minutes at 5-10 °C, maintain for 10-12 hours at 25-30 °C. The resulted protected peptide was subjected to treatment with tert-butylamine (1.0 mL), n-heptane (15 mL) in DMF. Filtered the precipitated solid and washed with water, hexane and DIPE to get H-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F- T(tBu/Oxa)-S(tBu)-D (OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(AEEAc- AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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 V.

Step-iii: Synthesis of crude Tirzepatide

Protected Tirzepatide (4.0 grams) was subjected to global cleavage in the presence of cocktail mixture (TFA, TIPS, water and DTT in the ratio of 87.5:5:5:2.5) at 10 tol5 °C for 2 hours under stirring. To this chilled MTBE was added and stirred for 2 hours, the precipitated solid was filtered and washed with DCM followed by DIPE to obtain crude Tirzepatide as solid.

Step-iv: Synthesis of Tirzepatide Crude Tirzepatide (10 grams) was dissolved in 0.5 M ammonium formate and loaded onto preparative C18 column (50x250 mm, 100 A0). The peptide was purified using a linear gradient of trifluoroacetic acid (0.1 %) and acetonitrile: methanol (8:1, 0.1% TFA) from 40 % to 90 % over 60 minutes. The pure fraction containing the Tirzepatide was pooled. The acetonitrile was evaporated, and the aqueous layer was lyophilized to give the Tirzepatide as white solid. The resulting peptide was analysed by RP-HPLC and confirmed by MALDI or LC-MS.

Yield: 1.0 gram.

Claims

We claim:

1. A process for the preparation of Tirzepatide compound of Formula I;

Formula I which comprises: i) condensation of H-G-P-S(tBu)-S(tBu)-G-A-P-P-P-S(tBu)-NH₂ (SEQ ID NO. 1) with Fmoc-A-F-V-Q(Trt)-W(Boc)-L-I-A-G-OH (SEQ ID NO. 2) in presence of coupling agent and solvent, followed by in-situ manner deprotection in presence of base to obtain 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 compound of Formula II; ii) condensing 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 compound of Formula II with Fmoc-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)- Eicosanedioic acid mono-t-butyl)-OH (SEQ ID NO. 3) in presence of coupling agent and solvent, followed by in-situ manner deprotection in presence of base to obtain H-Q(Trt)- K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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) condensation of H-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono- t-butyl)-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 with Fmoc-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)- K(Boc)-I-A-OH (SEQ ID NO. 4) in presence of coupling agent and solvent, followed by in-situ manner deprotection in presence of base to obtain H-S(tBu)-D(OtBu)-Y(tBu)- S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)- Eicosanedioic acid mono-t-butyl)-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 IV ; iv) condensation of H-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A- Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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 IV with Wl-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(W4)-OH (SEQ ID NO. 5) in presence of coupling agent and solvent, followed by in-situ manner deprotection in presence of base to obtain H-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(W4)-S(tBu)-D(OtBu)- Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a- OtBu)-Eicosanedioic acid mono-t-butyl)-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 (Protected Tirzepatide), where in W1 is Fmoc or CBZ and W4 is (tBu) or (Oxa); v) global deprotection of protected Tirzepatide using a reagent to obtain crude Tirzepatide or a pharmaceutically acceptable salt thereof; vi) purifying the crude Tirzepatide by preparative HPLC to obtain pure Tirzepatide or a pharmaceutically acceptable salt thereof.

2. A process for the preparation of Tirzepatide compound of Formula I;

Formula I which comprises: i) condensing H-G-P-S(tBu)-S(tBu)-G-A-P-P-P-S(tBu)-NH2 (SEQ ID NO. 1) with Fmoc- A-F-V-Q(Trt)-W(Boc)-L-I-A-G-OH (SEQ ID NO. 2) in presence of coupling agent and solvent, followed by in-situ manner deprotection in presence of base to obtain 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 compound of Formula II; ii) condensing 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 compound of Formula II with Fmoc-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)- Eicosanedioic acid mono-t-butyl)-OH (SEQ ID NO. 3) in presence of coupling agent and solvent, followed by in-situ manner deprotection in presence of base to obtain H-Q(Trt)- K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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) condensing H-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t- butyl)-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 with Fmoc-T(tBu)-F-T(tBu)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)- I-Aib-L-D(OtBu)-K(Boc)-I-A-OH (SEQ ID NO. 9) in presence of coupling agent and solvent, followed by in-situ manner deprotection in 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(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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 VI; iv) condensing 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(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t- butyl)-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 VI with W3-Y(tBu)-Aib-E(OtBu)-G-OH (SEQ ID NO. 10) in presence of coupling agent and solvent, followed by in-situ manner deprotection in 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(AEEAc-AEEAc-y-Glu (a- OtBu)-Eicosanedioic acid mono-t-butyl)-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 (Protected Tirzepatide), where in W3 is Fmoc, Boc or CBZ; v) global deprotection of protected Tirzepatide using a reagent to obtain crude Tirzepatide or a pharmaceutically acceptable salt thereof; vi) purifying the crude Tirzepatide by preparative HPLC to obtain pure Tirzepatide or a pharmaceutically acceptable salt thereof.

3. A process for the preparation of Tirzepatide compound of Formula I;

Formula I

Which comprises: i) condensing H-G-P-S(tBu)-S(tBu)-G-A-P-P-P-S(tBu)-NH2 (SEQ ID NO. 1) with Fmoc- Q(Trt)-K(W5)-A-F-V-Q(Trt)-W(Boc)-L-I-A-G-OH (SEQ ID NO. 6) in presence of coupling agent and solvent, followed by in-situ manner deprotection in presence of base to obtain H-Q(Trt)-K(W5)-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 VII, where in W5 is Dde or IVDde; ii) condensing H-Q(Trt)-K(W5)-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 VII with Fmoc-S(tBu)-D(OtBu)-Y(tBu)- S(tBu)-I-Aib-E-D(OtBu)-K(Boc)-I-A-OH (SEQ ID NO. 7) in presence of coupling agent and solvent, followed by in-situ manner deprotection in presence of base to obtain H-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(W5)-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 VIII; iii) condensing H-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)- K(W5)-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 VIII with CBZ-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(W4)-OH (SEQ ID NO. 8) in presence of coupling agent and solvent to obtain Cbz-Y(tBu)-Aib- E(OtBu)-G-T(tBu)-F-T(W4)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)- K(Boc)-I-A-Q(Trt)-K(Dde or IVDde)-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 IX, where in W4 is (tBu) or (Oxa); iv) deprotection of the Dde or IVDe group in presence of base and then it was coupled with NH₂ to obtain Cbz-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(W4)-S(tBu)-D(OtBu)-Y(tBu)- S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(NH₂)-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 X; v) deprotection of amino group in presence of base and then it was coupled with Fmoc- AEEAc-AEEAc-OH in presence of coupling agent and solvent followed by in-situ manner Fmoc removal to obtain Cbz-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(W4)-S(tBu)- D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(AEEAc-AEEAc)-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 XI; vi) condensation of Cbz-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(W4)-S(tBu)-D(OtBu)- Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(AEEAc-AEEAc)-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 XI with Fmoc-Glu-OtBu in presence of coupling agent and solvent followed by in-situ manner Fmoc removal to obtain Cbz-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F- T(W4)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)- K(AEEAc-AEEAc-y-Glu-OtBu)-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 XII; vii) condensation of Cbz-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(W4)-S(tBu)-D(OtBu)- Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(AEEAc-AEEAc-y-Glu(OtBu)- 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 XII with eicosane dioic acid mono-t-butyl ester in presence of coupling agent and solvent to obtain Cbz-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(W4)- S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(AEEAc- AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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 XIII; viii) deprotection of Cbz-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(W4)-S(tBu)-D(OtBu)- Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a- OtBu)-Eicosanedioic acid mono-t-butyl)-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 XIII in presence of base to obtain H-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(W4)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L- D(OtBu)-K(Boc)-I-A-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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 (Protected Tirzepatide); ix) global deprotection of protected Tirzepatide using a reagent to obtain crude Tirzepatide or a pharmaceutically acceptable salt thereof; x) purifying the crude Tirzepatide by preparative HPLC to obtain pure Tirzepatide or a pharmaceutically acceptable salt thereof.

4. A process for the preparation of Tirzepatide compound of Formula I;

Formula I Which comprises: i) condensation of H-G-P-S(tBu)-S(tBu)-G-A-P-P-P-S(tBu)-NH₂ (SEQ ID NO. 1) with Cbz-Q(Trt)-K(W5)-A-F-V-Q(Trt)-W(Boc)-L-I-A-G-OH (SEQ ID NO. 6) in presence of coupling agent and solvent, followed by in-situ manner deprotection in presence of base to obtain Cbz-Q(Trt)-K(W5)-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 XIV, where in W5 is Dde or IVDde; ii) deprotection of the Dde or IVDe group in presence of base and then it was coupled with NH2 to obtain Cbz-Q(Trt)-K(NH₂)-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 XV ; iii) deprotection of amino group in presence of base and then it was coupled with Fmoc- AEEAc-AEEAc-OH in presence of coupling agent and solvent, followed by in-situ manner Fmoc removal to obtain Cbz-Q(Trt)-K(AEEAc-AEEAc)-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 XVI; iv) condensation of Cbz-Q(Trt)-K(AEEAc-AEEAc)-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 XVI with Fmoc-Glu-OtBu in presence of coupling agent and solvent, followed by in-situ manner Fmoc removal to obtain Cbz-Q(Trt)-K(AEEAc-AEEAc-y-Glu-OtBu)-A-F-V-Q(Trt)-W(Boc)-E-I-A-G-G- P-S(tBu)-S(tBu)-G-A-P-P-P-S(tBu)-NH₂ compound of Formula XVII; v) condensation of Cbz-Q(Trt)-K(AEEAc-AEEAc-y-Glu-OtBu)-A-F-V-Q(Trt)-W(Boc)-E- I-A-G-G-P-S(tBu)-S(tBu)-G-A-P-P-P-S(tBu)-NH₂ compound of Formula XVII with eicosane dioic acid mono-t-butyl ester in presence of coupling agent and solvent, followed by in-situ manner Z group removal to obtain H-Q(Trt)-K(AEEAc-AEEAc-y- Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-A-F-V-Q(Trt)-W(Boc)-E-I-A-G-G-P- S(tBu)-S(tBu)-G-A-P-P-P-S(tBu)-NH₂ compound of Formula XVIII; vi) condensation of H-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono- t-butyl)-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 XVIII with Fmoc-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-E- D(OtBu)-K(Boc)-I-A-OH (SEQ ID NO. 7) in presence of coupling agent and solvent, followed by in-situ manner deprotection in presence of base to obtain H-S(tBu)-D(OtBu)- Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(AEEAc-AEEAc-Y-Glu (a- OtBu)-Eicosanedioic acid mono-t-butyl)-A-F-V-Q(Trt)-W(Boc)-E-I-A-G-G-P-S(tBu)- S(tBu)-G-A-P-P-P-S(tBu)-NH₂ compound of Formula XIX; vii) condensation of H-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A- Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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 XIX with W3-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(W4)-OH (SEQ ID NO. 8) in presence of coupling agent and solvent, followed by in-situ manner deprotection in presence of base to obtain H-Y(tBu)-Aib-E(OtBu)-G-T(tBu)-F-T(W4)-S(tBu)-D(OtBu)- Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a- OtBu)-Eicosanedioic acid mono-t-butyl)-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 (Protected Tirzepatide), where in W4 is (tBu) or (Oxa); viii) global deprotection of protected Tirzepatide using a reagent to obtain crude Tirzepatide or a pharmaceutically acceptable salt thereof; ix) purifying the crude Tirzepatide by preparative HPLC to obtain pure Tirzepatide or a pharmaceutically acceptable salt thereof.

5. A process for the preparation of Tirzepatide compound of Formula I;

Formula I

Which comprises: i) condensation of W2-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 (a- OtBu)-Eicosanedioic acid mono-t-butyl)-OH (SEQ ID NO. 11) 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 (SEQ ID NO. 12) in presence of coupling agent and solvent, followed by in-situ manner deprotection in presence of base to obtain (Protected Tirzepatide), where in W2 is Fmoc or Boc; ii) global deprotection of protected Tirzepatide using a reagent to obtain crude Tirzepatide or a pharmaceutically acceptable salt thereof; iii) purifying the crude Tirzepatide by preparative HPLC to obtain pure Tirzepatide or pharmaceutically acceptable salt thereof. A process for the preparation of Tirzepatide of Formula I;

Formula I which comprises i. condensation 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 (SEQ ID NO. 12) with Fmoc-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)- Eicosanedioic acid mono-t-butyl)-OH (SEQ ID NO. 3) in presence of coupling agent and solvent, followed by in-situ manner deprotection in presence of base to obtain H- Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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; ii. condensation of H-Q(Trt)-K(AEEAc-AEEAc-y-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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 with W2-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-OH (SEQ ID NO. 13) in presence of coupling agent and solvent, followed by in-situ manner deprotection in 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(AEEAc-AEEAc-y- Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl)-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 (Protected Tirzepatide), where in W2 is Fmoc or Boc; iii. global deprotection of protected Tirzepatide using a reagent to obtain crude Tirzepatide or a pharmaceutically acceptable salt thereof; iv. sequential deprotection and coupling of Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-Ser(Oxa)-OH to the obtained resin in step-iii) in presence of a coupling agent and solvent to obtained 8 amino acid peptide resin; v. purifying the crude Tirzepatide by preparative HPLC to obtain pure Tirzepatide or a pharmaceutically acceptable salt thereof.

7. A compound of SEQ ID NO. 1 to 14 is

8. A compound of Formulae II to Formulae XX

9. The process as claimed in claim 1 to 6, wherein said base is selected from group consisting of potassium carbonate, lithium carbonate, sodium carbonate, sodium hydroxide, potassium hydroxide, diisopropyl amine, N,N-diisopropyl ethylamine, triethylamine, tertiary butyl amine, dimethylamine, tri methyl amine, isopropyl ethylamine, pyridine, piperidine, N- methyl morpholine or a mixture thereof.

10. The process as claimed in claim 1 to 6, wherein said coupling agent is selected from group consisting of Dicyclohexyl carbodiimide (DCC), di isopropyl carbodiimide (DIC), 1 -hydroxy benzotriazole (HOBt), ethyl-2-cyano-2-(hydroxy amino) acetate (Oxyma pure), 1 -(dimethyl aminopropyl)-3-ethylcarbodiimide hydrochloride (EDC HC1) or a mixture thereof.

11. The process as claimed in claim 1 to 6, wherein said solvent is selected from group consisting dimethylacetamide, dimethylformamide, formamide, N-Methylformamide, N-Methyl-2- pyrrolidone, Dimethylacetamide, 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, acetonitrile, propionitrile, butyronitrile, isobutyronitrile, acetone, ethyl methyl ketone, methyl isobutyl ketone, diethyl ketone, pentane, n-heptane, water or a mixture thereof.