DESC:Title of the Invention
An improved process for the preparation of Tirzepatide by a linear synthesis.

Field of the Invention

The present invention relates to an improved process for the preparation of Tirzepatide by a linear synthesis having the chemical structural of Formula I.

The present invention also relates to novel intermediates of Formulae II, III and its process of preparation, which are useful in the preparation of Tirzepatide.

Formula II: Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(AEEAc-AEEAc-?-Glu-OtBu-19-carboxynonadecanoyl mono t-butylester)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Rink amide MBHA resin/ Sieber Amide resin.

Formula III: Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(Oxa)-Asp(OtBu)-Tyr(tBu)-Ser(Oxa)-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(AEEAc-AEEAc-?-Glu-OtBu-19-carboxynonadecanoyl mono t-butylester)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(Oxa)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Rink amide MBHA resin/ Sieber Amide resin.

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-1,10,19,24-tetraoxo-3,6,12,15-tetraoxa- 9, 18, 23-triazadotetracont-1-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(Linker)-Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2. The molecular formula is C225H348N48O68. The structural Formula is:

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 A1, WO 2021/158444 A1, CN 112110981 A, CN 112661815 A, WO 2022079639 A1 and WO 2021260530 A1.

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

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 by a linear synthesis.

The present invention provides a cost effective, novel and an efficient process for the preparation of Tirzepatide by employing linear synthesis, the final product is prepared by sequential condensation of single amino acids on solid support to obtained final peptide with good yields and purity.

In one embodiment, the present invention relates to an improved process for the preparation of Tirzepatide by using linear synthesis refers to a process whereby the final product or an intermediate thereof is prepared by sequential transformations of a single starting material. Typically, in a linear synthesis, the final product is prepared by sequential condensation of single amino acids, optionally further suitably side chain protected amino acids to build the final peptide with target sequence or dipeptides comprising pseudoproline dipeptides, in a linear fashion, with the proviso that at the unique.

The present invention provides a linear solid phase synthesis of Tirzepatide of Formula I by sequential coupling of single Fmoc protected amino acids.

which comprises:
i) anchoring Fmoc-Ser(tBu)-OH to a resin in presence of a base;
ii) selective deprotection of amino acid using a base;
iii) coupling of Fmoc-Pro-OH to a resin obtained in step-ii) in presence of coupling agent in a solvent to obtain dipeptide resin;
iv) sequential deprotection and coupling of Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-Lys(Linker)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Aib-OH, Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Aib-OH and Fmoc-Tyr(tBu)-OH to the obtained resin in step-iii) in presence of a coupling agent and solvent to obtained protected Tirzepatide of Formula II;
v) cleaving the protected Tirzepatide using a reagent to obtain crude Tirzepatide;
vi) purifying the crude Tirzepatide by preparative HPLC to obtain pure Tirzepatide.

In another embodiment, the present invention relates to novel intermediate of Boc- Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(AEEAc-AEEAc-?-Glu-OtBu-19-carboxynonadecanoyl mono t-butylester)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Rink amide MBHA resin/ Sieber Amide resin compound of Formula II.

In another embodiment, the present invention provides an improved process for the preparation of Tirzepatide of Formula I by employing dipeptides comprising pseudoproline dipeptides in linear synthesis,

which comprises:
i) anchoring Fmoc-Ser(tBu)-OH to a resin in presence of a base;
ii) selective deprotection of amino acid using a base;
iii) coupling of Fmoc-Pro-OH to a resin obtained in step-ii) in presence of coupling agent in a solvent to obtain dipeptide resin;
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) sequential deprotection and coupling of Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-Lys(Linker)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Aib-OH, Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH; optionally Fmoc-Tyr(tBu)-Ser(Oxa)-OH to the obtained resin in step-iv) in presence of a coupling agent and solvent to obtained 30 amino acid peptide resin;
vi) sequential deprotection and coupling of Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH; optionally Fmoc-Thr(tBu)-Ser(Oxa)-OH to the obtained resin in step-v) in presence of a coupling agent and solvent to obtain 33 amino acid peptide resin;
vii) sequential deprotection and coupling of Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Aib-OH and Fmoc-Tyr(tBu)-OH to the obtained resin in step-vi) in presence of a coupling agent and solvent to obtained protected Tirzepatide of Formula III;
viii) cleaving the protected Tirzepatide using a reagent to obtain crude Tirzepatide;
ix) purifying the crude Tirzepatide by preparative HPLC to obtain pure Tirzepatide.

In another embodiment, the present invention relates to novel intermediate of Boc- Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(Oxa)-Asp(OtBu)-Tyr(tBu)-Ser(Oxa)-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(AEEAc-AEEAc-?-Glu-OtBu-19-carboxynonadecanoyl mono t-butylester)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(Oxa)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Rink amide MBHA resin/ Sieber Amide resin compound of Formula III.

Detailed Description of the Invention

Unless otherwise stated, the following terms used in the specification and claims have the meanings given below:

The following three letter amino acid abbreviations are used throughout the text:
Alanine: (Ala) A Arginine: (Arg) R
Asparagine: (Asn) N Aspartic acid: (Asp) D
Cysteine: (Cys) C Glutamine: (Gln) Q
Glutamic acid: (Glu) E Glycine: (Gly) G
Histidine: (His) H Isoleucine: (Ile) 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: (Val) 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

Solid phase peptide synthesis is carried out on an insoluble polymer which is acid sensitive. Acid sensitive resin selected from the group consisting of Rink amide MBHA resin, sieber amide resin, Rink amide AM resin, MBHA and rink acid resin. Preferably using Rink amide MBHA 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 Isopropyl alcohol (IPA) or a mixture.
Coupling of amino acid to a resin is carried out in presence of a base. The base is organic or inorganic base. The inorganic base is selected from the group consisting of potassium carbonate, lithium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, sodium hydroxide, potassium hydroxide, ammonium hydroxide and mixture thereof; the organic base is selected from the group consisting of diisopropyl amine, N, N-diisopropyl ethylamine, triethylamine, tertiary butyl amine, dimethylamine, tri methyl amine, isopropyl ethylamine, pyridine, piperidine, N-methyl morpholine or a mixture thereof.
Solvents are used throughout the invention is selected from the group consisting of hydrocarbon solvents such as 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, 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 in the reaction can be selected from the group consisting of Ethylcyano (hydroxyimino)acetate-2)-tri-(1-pyrrolidinyl)-Phosphonium hexa fluorophosphate (PyOxim), ethyl-2-cyano-2-(hydroxy amino) acetate (Oxyma pure), O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), diisopropyl carbodiimide (DIC), 1,3-dicyclohexylcabodiimide (DCC), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), 1-(dimethyl aminopropyl)-3-ethylcarbodiimide hydrochloride (EDC HCl), O-(benzotriazol-1-yl)-1,1,3,3-tetra methyluronium hexafluorophosphate (HBTU), 1-Hydroxybenzotriazole (HOBt), Isopropyl chloro formate (IPCF), Benzotriazol-1-yl-oxy-tris(dimethyl-amino)-phosphonium hexa fluorophosphate (BOP), benzotriazole-1-yloxytri(pyrrolidino)phosphonium hexa fluoro phosphate (PyBOP), N,N-bis-(2-oxo-3-oxazolidinyl)phosphonic dichloride (BOP-Cl), bromotri(pyrrolidino)phosphonium hexa fluoro phosphate (PyBrOP), O-(6-Chloro-1-hydrocibenzotriazol-1-yl)-1,1,3,3-tetramethyl uranium tetra fluoroborate (TCTU), chlorotri (pyrrolidino)phosphonium hexafluorophosphate (PyClOP), Ethyl 1,2-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 a mixture.

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 term “linker” as used herein AEEA-AEEA-?-Glu-Eicosanedioic acid (or) AEEA-AEEA-?-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl (or) AEEAc-AEEAc-?-Glu-OtBu-19-carboxynonadecanoyl mono t-butylester.

The term “peptide” as used herein includes the peptide as well as pharmaceutically acceptable salts of the peptide. Peptide are prepared by using solid phase peptide synthesis through linear approach.

Typically, the single amino acids are optionally side-chain protected as well as N-terminal protected with the usual protecting groups for peptide synthesis. Preferably, the N-terminal protecting groups are Fmoc, Boc or Cbz, and more preferably Fmoc or Boc. The condensation(s) can be carried out through solid phase synthesis or in liquid phase or a combination of both, followed by purification on reverse phase HPLC, freeze drying and isolation to get pure Tirzepatide.

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

The coupling reactions may be monitored by kaiser test, 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.

The present disclosure relates to a process for the linear synthesis of a Tirzepatide thereof, the process comprising the stepwise addition of selected amino acids and pseduoprolin di-peptide blocks at appropriate positions in the targeted peptide sequence.

Advantages for using pseudo proline dipeptides to incorporation of pseudo proline blocks in growing peptide sequence enhances the coupling efficiency by inhibiting/ reducing the aggregation between the peptide strands. It also increases solubility of the peptide fragments there by increase the coupling efficiency with incoming amino acid & peptide fragment.

In one embodiment, the present invention relates a novel process for the preparation of Tirzepatide by employing linear synthesis in a required sequence, deprotection and condensing them in solution phase, followed by purification to get Tirzepatide. The schematic description of the process is as shown in Scheme-I.

In step-i), Rink amide MBHA resin was taken in a SPPS reactor and swollen by adding of dimethylformamide, deprotecting the Fmoc-resin in presence of a base, preferably using 20% piperidine in dimethylformamide. The first amino acid Fmoc-Ser(tBu)-OH and Oxyma & DIC was added to the resulting reaction mixture in dimethylformamide.

In step ii), deprotecting the Fmoc group in presence of a base, preferably using 20% piperidine in dimethylformamide (DMF).

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 1 to 4 hours, preferably for a period of 2 to 4 hours.

In step iii), condensation of peptide resin obtained in step-ii) with Fmoc-Pro-OH in presence of coupling agent.

In step iv), sequential deprotection and coupling of Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-Lys(Linker)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Aib-OH, Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Aib-OH and Fmoc-Tyr(tBu)-OH to the obtained resin in step-iii) in presence of a coupling agent and solvent to obtained protected Tirzepatide 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 1 to 4 hours, preferably for a period of 2 to 4 hours.

In step v), cleaving the protected Tirzepatide using a reagent to obtain crude Tirzepatide. Diisopropyl ether was added and stirred for 2 hours to obtained solid was filtered and washed with dichloromethane, diisopropyl ether to get the 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 to 7 hours, preferably for a period of 3 to 6 hours.

In step vi), purifying the crude Tirzepatide by preparative HPLC to obtain pure Tirzepatide.

The coupling agent used in these steps are DIC, Oxyma pure in DMF. Deprotection carried out using 20 % of piperidine in Dimethylformamide.

Reagent used in global deprotection is selected from the group consisting of TFA, TIPS, Water, DTT, Thioanisole, EDT, DMS, cresol, phenol, thiocresol, ammonium iodide, 2,2'-(ethylene dioxy)diethane or its mixture.

The cleavage cocktail mixture consisting of TFA/TIPS/Water/DTT range from 70%/2.5%/2.5%/1% to 95%/10%/10%/5%, preferably cocktail mixture is 90%/5%/5%/2.5%.

The Peptide was purified using a linear gradient of aqueous TFA (0.1%) and acetonitrile: methanol (8:1, 0.1% TFA) from 40% to 90% over 60 minutes.
The reaction was monitored by TNBS/ Chloranil / Kaiser test.
In another embodiment, the present invention relates a novel process for the preparation of Tirzepatide by employing dipeptides comprising pseudoproline dipeptides in linear synthesis in a required sequence, deprotection and condensing them in solution phase, followed by purification to get Tirzepatide. The schematic description of the process is as shown in Scheme-II.

In step-i), Rink amide MBHA resin was taken in a SPPS reactor and swollen by adding of dimethylformamide, deprotecting the Fmoc-resin in presence of a base, preferably using 20% piperidine in dimethylformamide. The first amino acid Fmoc-Ser(tBu)-OH and Oxyma & DIC was added to the resulting reaction mixture in dimethylformamide.

In step ii), deprotecting the Fmoc group in presence of a base, preferably using 20% piperidine in dimethylformamide (DMF);

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 1to 4 hours, preferably for a period of 2 to 4 hours.

In step iii), condensation of peptide resin obtained in step-ii) with Fmoc-Pro-OH in presence of coupling agent;

In step 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;

In step v), sequential deprotection and coupling of Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-Lys(Linker)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Aib-OH, Fmoc-Ile-OH or Fmoc-Ile-Aib-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH; optionally Fmoc-Tyr(tBu)-Ser(Oxa)-OH to the obtained resin in step-iv) in presence of a coupling agent and solvent to obtained 30 amino acid peptide resin;

In step vi), sequential deprotection and coupling of Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH; optionally Fmoc-Thr(tBu)-Ser(Oxa)-OH to the obtained resin in step-v) in presence of a coupling agent and solvent to obtain 33 amino acid peptide resin;

In step vii), sequential deprotection and coupling of Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Aib-OH and Fmoc-Tyr(tBu)-OH to the obtained resin in step-vi) in presence of a coupling agent and solvent to obtained protected Tirzepatide 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 1 to 4 hours, preferably for a period of 2 to 4 hours.

In step viii), cleaving the protected Tirzepatide using a reagent to obtain crude Tirzepatide. Diisopropyl ether was added and stirred for 2 hours to obtained solid was filtered and washed with dichloromethane, diisopropyl ether to get the 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 to 7 hours, preferably for a period of 3 to 6 hours.

In step ix), purifying the crude Tirzepatide by preparative HPLC to obtain pure Tirzepatide.

The coupling agent used in these steps are DIC, Oxyma pure in DMF. Deprotection carried out using 20 % of piperidine in Dimethylformamide.

Reagent used in partial deprotection is selected from the group consisting of TFA, TIPS, Water, DTT, Thioanisole, EDT, DMS, cresol, phenol, thiocresol, ammonium iodide, 2,2'-(ethylene dioxy)diethane or its mixture.

The cleavage cocktail mixture consisting of TFA/TIPS/Water/DTT range from 70%/2.5%/2.5%/1% to 95%/10%/10%/5%, preferably cocktail mixture is 90%/5%/5%/2.5%.

The Peptide was purified using a linear gradient of aqueous TFA (0.1%) and acetonitrile: methanol (8:1, 0.1% TFA) from 40% to 90% over 60 minutes.

The reaction was monitored by TNBS/ Chloranil / Kaiser test.

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 (50×250 mm, 10 µm)
Mobile phase-A: Trifluoro 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.

S. No Time Flow (mL/min) Mobile Phase A% Mobile Phase B%
1 0.01 60 95 5
2 10 60 75 25
3 150 60 40 60
4 200 60 0 100
5 300 60 0 100

Collect the fractions as 25 mL/vial

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.

S. No Time Flow (mL/min) Mobile Phase A% Mobile Phase B%
1 0.01 50 95 5
2 10 50 75 25
3 150 50 50 50
4 200 50 0 100
5 300 50 0 100

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: Linear solid phase peptide synthesis of Tirzepatide
Step I: Rink Amide MBHA resin (10 grams) was taken in a SPPS reactor and dimethylformamide (100 mL) was added and allowed it to swell for 10 minutes. Fmoc-resin was deblocked by treatment with 20 % piperidine in DMF solution. Resin bed wash with dimethylformamide, isopropyl alcohol, dichloromethane and finally wash with dimethylformamide.
Step II: The first amino acid of Fmoc-Ser(tBu)-OH (3 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with oxyma (3.41 mL) and N,N'-diisopropylcarbodiimide (3.71 mL) and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained from step (I) and stirred for 2-4 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step III: Fmoc-Pro-OH (7.5 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.19 grams) and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step (II) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step IV: Fmoc-Pro-OH (7.5 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step (III) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step V: Fmoc-Pro-OH (7.5 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step (IV) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step VI: Fmoc-Ala-OH (7.0 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step (V) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step VII: Fmoc-Gly-OH (6.6 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step (VI) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step VIII: Fmoc-Ser(tBu)-OH (8.6 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step (VII) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step IX: Fmoc-Ser(tBu)-OH (8.6 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step (VIII) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step X: Fmoc-Pro-OH (7.5 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step (X) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step XI: Fmoc-Gly-OH (6.6 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step (X) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step XII: Fmoc-Gly-OH (6.6 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step (XI) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step XIII: Fmoc-Ala-OH (7.0 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step (XII) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step XIV: Fmoc-Ile-OH (7.9 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step (XIII) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step XV: Fmoc-Leu-OH (7.9 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step (XIV) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step XVI: Fmoc-Trp(Boc)-OH (11.8 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step (XV) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step XVII: Fmoc-Gln(Trt)-OH (13.7 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step (XVI) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step XVIII: Fmoc-Val-OH (7.6 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1grams) and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step (XVII) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step XIX: Fmoc-Phe-OH (8.7 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step (XVIII) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step XX: Fmoc-Ala-OH (7.0 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1grams) and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step (XIX) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step XXI: Fmoc-Gln(Trt)-Lys(Linker)-OH (11.96grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1grams) and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step (XX) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step XXII: Fmoc-Ala-OH (7.0 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step (XXI) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step XXIII: Fmoc-Ile-OH (7.9 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step (XXII) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step XXIV: Fmoc-Lys(Boc)-OH (10.5 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the Loom temperature. It was added to the resin obtained in step (XXIII) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step XXV: Fmoc-Asp(OtBu)-OH (9.2 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the Loom temperature. It was added to the resin obtained in step (XXIV) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step XXVI: Fmoc-Leu-OH (7.9 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the Loom temperature. It was added to the resin obtained in step (XXV) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step XXVII: Fmoc-Aib-OH (7.3 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the Loom temperature. It was added to the resin obtained in step (XXVI) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step XXVIII: Fmoc-Ile-OH (7.9 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the Loom temperature. It was added to the resin obtained in step (XXVII) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step XXIX: Fmoc-Ser(tBu)-OH (8.6 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the Loom temperature. It was added to the resin obtained in step (XXVIII) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step XXX: Fmoc-Tyr(tBu)-OH (10.3 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the Loom temperature. It was added to the resin obtained in step (XXIX) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step XXXI: Fmoc-Asp(OtBu)-OH (9.2 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the Loom temperature. It was added to the resin obtained in step (XXX) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step XXXII: Fmoc-Ser(tBu)-OH (9.6 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the Loom temperature. It was added to the resin obtained in step (XXXI) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step XXXIII: Fmoc-Thr(tBu)-OH (90 grams) was dissolved in dimethylformamide (100 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.48 mL) and oxyma (3.2 grams) and stirred for 5-10 minutes at the Loom temperature. It was added to the resin obtained in step (XXXII) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (600 mL), isopropanol (200 mL) and dichloromethane (200 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (150 mL) for 10 minutes and washed with dimethylformamide (400 mL).
Step XXXIV: Fmoc-Phe-OH (8.7 grams) was dissolved in dimethylformamide (100 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.48 mL) and oxyma (3.2 grams) and stirred for 5-10 minutes at the Loom temperature. It was added to the resin obtained in step (XXXIII) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (600 mL), isopropanol (200 mL) and dichloromethane (200 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (150 mL) for 10 minutes and washed with dimethylformamide (400 mL).
Step XXXV: Fmoc-Thr(tBu)-OH (9.0 grams) was dissolved in dimethylformamide (100 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.48 mL) and oxyma (3.2 grams) and stirred for 5-10 minutes at the Loom temperature. It was added to the resin obtained in step (XXXIV) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (600 mL), isopropanol (200 mL) and dichloromethane (200 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (150 mL) for 10 minutes and washed with dimethylformamide (400 mL).
Step XXXVI: Fmoc-Gly-OH (6.6 grams) was dissolved in dimethylformamide (100 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.2 grams) and stirred for 5-10 minutes at the Loom temperature. It was added to the resin obtained in step (XXXV) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (600 mL), isopropanol (200 mL) and dichloromethane (200 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (150 mL) for 10 minutes and washed with dimethylformamide (400 mL).
Step XXXVII: Fmoc-Glu(OtBu)-OH (9.5 grams) was dissolved in dimethylformamide (100 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.2 grams) and stirred for 5-10 minutes at the Loom temperature. It was added to the resin obtained in step (XXXVI) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (600 mL), isopropanol (200 mL) and dichloromethane (200 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (150 mL) for 10 minutes and washed with dimethylformamide (400 mL).
Step XXXVIII: Fmoc-Aib-OH (7.3 grams) was dissolved in dimethylformamide (100 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.2 grams) and stirred for 5-10 minutes at the Loom temperature. It was added to the resin obtained in step (XXXVII) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (600 mL), isopropanol (200 mL) and dichloromethane (200 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (150 mL) for 10 minutes and washed with dimethylformamide (400 mL).
Step XXXIX: Fmoc-Tyr(tBu)-OH (10.35 grams) was dissolved in dimethylformamide (100 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.2 grams) and stirred for 5-10 minutes at the Loom temperature. It was added to the resin obtained in step (XXXVIII) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (600 mL), isopropanol (200 mL) and dichloromethane (200 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (150 mL) for 10 minutes and washed with dimethylformamide (400 mL).
Step XL: Coupling of the last amino acid and resin was washed with DMF (400 mLx2), Isopropanol (400 mL) and MDC (400 mL) then dried and washed with Hexane (400 mL) followed by dried under vacuum at 25-30 °C for 6-8 hours to obtain Protected Tirzepatide of Formula II.
Step XLI: Selective cleavage of Rink amide MHBA resin or Fmoc-Sieber amide resin and protected amine from Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(AEEAc-AEEAc-?-Glu-OtBu-19-carboxynonadecanoyl mono t-butylester)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Rink amide MBHA resin or Fmoc-Sieber amide resin by using mixture of TFA/DTT/Water/Tips [85%/5 %/5%/5%]. The reaction mass was stirred for 3-6 hours at 10-15°C then chilled DIPE was added to the reaction mixture and stirred for 2 hours. The obtained solid was filtered and washed with DCM, DIPE to get the crude Tirzepatide.
Step XLII: Crude Tirzepatide (20 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 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: 2.0 grams.

Example 2: Pseudoproline solid phase peptide synthesis of Tirzepatide
Step I: Rink Amide MBHA resin (10 grams) was taken in a SPPS reactor and dimethylformamide (100 mL) was added and allowed it to swell for 10 minutes. Fmoc-resin was deblocked by treatment with 20 % piperidine in DMF solution. Resin bed wash with dimethylformamide, isopropyl alcohol, dichloromethane and finally wash with dimethylformamide.
Step II: The first amino acid of Fmoc-Ser(tBu)-OH (3 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with oxyma (3.41 mL) and N,N'-diisopropylcarbodiimide (3.71 mL) and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained from step (I) and stirred for 2-4 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step III: Fmoc-Pro-OH (7.5 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.19 grams) and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step (II) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step IV: Fmoc-Pro-OH (7.5 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step (III) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step V: Fmoc-Pro-OH (7.5 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step (IV) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step VI: Fmoc-Ala-OH (7.0 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step (V) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step VII: Fmoc-Gly-OH (6.6 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step (VI) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step VIII: Fmoc-Ser(tBu)-Ser(Oxa)-OH (8.6 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step (VII) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step IX: Fmoc-Pro-OH (7.5 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step (VIII) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step X: Fmoc-Gly-OH (6.6 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step (IX) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step XI: Fmoc-Gly-OH (6.6 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step (X) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step XII: Fmoc-Ala-OH (7.0 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step (XI) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step XIII: Fmoc-Ile-OH (7.9 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step (XII) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step XIV: Fmoc-Leu-OH (7.9 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step (XIII) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step XV: Fmoc-Trp(Boc)-OH (11.8 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step (XIV) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step XVI: Fmoc-Gln(Trt)-OH (13.7 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step (XV) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step XVII: Fmoc-Val-OH (7.6 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1grams) and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step (XVI) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step XVIII: Fmoc-Phe-OH (8.7 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step (XVII) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step XIX: Fmoc-Ala-OH (7.0 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1grams) and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step (XVIII) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step XX: Fmoc-Gln(Trt)-Lys(Linker)-OH (11.96 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1grams) and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step (XIX) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step XXI: Fmoc-Ala-OH (7.0 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step (XX) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step XXII: Fmoc-Ile-OH (7.9 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step (XXI) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step XXIII: Fmoc-Lys(Boc)-OH (10.5 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the Loom temperature. It was added to the resin obtained in step (XXII) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step XXIV: Fmoc-Asp(OtBu)-OH (9.2 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the Loom temperature. It was added to the resin obtained in step (XXIII) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step XXV: Fmoc-Leu-OH (7.9 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the Loom temperature. It was added to the resin obtained in step (XXIV) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step XXVI: Fmoc-Aib-OH (7.3 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the Loom temperature. It was added to the resin obtained in step (XXV) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step XXVII: Fmoc-Ile-OH (7.9 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the Loom temperature. It was added to the resin obtained in step (XXVI) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step XXVIII: Fmoc-Tyr(tBu)-Ser(Oxa)-OH (13.2 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the Loom temperature. It was added to the resin obtained in step (XXVII) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step XXIX: Fmoc-Asp(OtBu)-OH (9.2 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the Loom temperature. It was added to the resin obtained in step (XXVIII) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step XXX: Fmoc-Thr(tBu)-Ser(Oxa)-OH (11.8 grams) was dissolved in dimethylformamide (70 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.1 grams) and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step (XXIX) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (70 mL), isopropanol (70 mL) and dichloromethane (70 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (70 mL).
Step XXXI: Fmoc-Phe-OH (8.7 grams) was dissolved in dimethylformamide (100 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.48 mL) and oxyma (3.2 grams) and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step (XXX) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (600 mL), isopropanol (200 mL) and dichloromethane (200 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (150 mL) for 10 minutes and washed with dimethylformamide (400 mL).
Step XXXII: Fmoc-Thr(tBu)-OH (9.0 grams) was dissolved in dimethylformamide (100 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.48 mL) and oxyma (3.2 grams) and stirred for 5-10 minutes at the Loom temperature. It was added to the resin obtained in step (XXXI) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (600 mL), isopropanol (200 mL) and dichloromethane (200 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (150 mL) for 10 minutes and washed with dimethylformamide (400 mL).
Step XXXIII: Fmoc-Gly-OH (6.6 grams) was dissolved in dimethylformamide (100 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.2 grams) and stirred for 5-10 minutes at the Loom temperature. It was added to the resin obtained in step (XXXII) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (600 mL), isopropanol (200 mL) and dichloromethane (200 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (150 mL) for 10 minutes and washed with dimethylformamide (400 mL).
Step XXXIV: Fmoc-Glu(OtBu)-OH (9.5 grams) was dissolved in dimethylformamide (100 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.2 grams) and stirred for 5-10 minutes at the Loom temperature. It was added to the resin obtained in step (XXXIII) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (600 mL), isopropanol (200 mL) and dichloromethane (200 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (150 mL) for 10 minutes and washed with dimethylformamide (400 mL).
Step XXXV: Fmoc-Aib-OH (7.3 grams) was dissolved in dimethylformamide (100 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.2 grams) and stirred for 5-10 minutes at the Loom temperature. It was added to the resin obtained in step (XXXIV) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (600 mL), isopropanol (200 mL) and dichloromethane (200 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (150 mL) for 10 minutes and washed with dimethylformamide (400 mL).
Step XXXVI: Fmoc-Tyr(tBu)-OH (10.35 grams) was dissolved in dimethylformamide (100 mL) and stirred for 10 minutes then activated with N,N'-diisopropylcarbodiimide (3.4 mL) and oxyma (3.2 grams) and stirred for 5-10 minutes at the Loom temperature. It was added to the resin obtained in step (XXXV) and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (600 mL), isopropanol (200 mL) and dichloromethane (200 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (150 mL) for 10 minutes and washed with dimethylformamide (400 mL).
Step XXXVII: Coupling of the last amino acid the resin was washed with DMF (400 mLx2), Isopropanol (400 mL) and MDC (400 mL) then dried and washed with Hexane (400 mL) and dried under vacuum at 25-30°C for 6-8 hours to obtain Protected Tirzepatide of Formula III.
Step XXXVIII: Selective cleavage of Rink amide MHBA resin or Fmoc-Sieber amide resin and protected amine from Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(AEEAc-AEEAc-?-Glu-OtBu-19-carboxynonadecanoyl mono t-butylester)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Rink amide MBHA resin by using a mixture of TFA/DTT/Water/Tips [85%/5 %/5%/5%]. The reaction mass was stirred for 3-6 hours at 10-15°C then chilled DIPE was added to the reaction mixture and stirred for 2 hours. The obtained solid was filtered and washed with DCM, DIPE to obtain crude Tirzepatide.
Step XXXIX: Crude Tirzepatide (20 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 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: 2.1 grams.
,CLAIMS:We claim:

1. An improved process for linear solid phase synthesis of Tirzepatide of Formula I by sequential coupling of single Fmoc protected amino acids;

which comprises:
i) anchoring Fmoc-Ser(tBu)-OH to a resin in presence of a base;
ii) selective deprotection of amino acid using a base;
iii) coupling of Fmoc-Pro-OH to a resin obtained in step-ii) in presence of coupling agent in a solvent to obtain dipeptide resin;
iv) sequential deprotection and coupling of Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-Lys(Linker)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Aib-OH, Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Aib-OH and Fmoc-Tyr(tBu)-OH to the obtained resin in step-iii) in presence of a coupling agent and solvent to obtained protected Tirzepatide of Formula II;
v) cleaving the protected Tirzepatide using a reagent to obtain crude Tirzepatide;
vi) purifying the crude Tirzepatide by preparative HPLC to obtain pure Tirzepatide.
2. A compound of Formula II is Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(AEEAc-AEEAc-?-Glu-OtBu-19-carboxynonadecanoyl mono t-butylester)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Rink amide MBHA resin/ Sieber Amide resin.

3. An improved process for the preparation of Tirzepatide of Formula I by employing dipeptides comprising pseudoproline dipeptides in linear synthesis;

which comprises
i. anchoring Fmoc-Ser(tBu)-OH to a resin in presence of a base;
ii. selective deprotection of amino acid using a base;
iii. coupling of Fmoc-Pro-OH to a resin obtained in step-ii) in presence of coupling agent in a solvent to obtain dipeptide resin;
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. sequential deprotection and coupling of Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-Lys(Linker)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Aib-OH, Fmoc-Ile-OH; optionally Fmoc-Ile-Aib-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH; optionally Fmoc-Tyr(tBu)-Ser(Oxa)-OH to the obtained resin in step-iv) in presence of a coupling agent and solvent to obtained 30 amino acid peptide resin;
vi. sequential deprotection and Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH; optionally Fmoc-Thr(tBu)-Ser(Oxa)-OH to the obtained resin in step-v) in presence of a coupling agent and solvent to obtain 33 amino acid peptide resin;
vii. sequential deprotection and coupling of Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Aib-OH and Fmoc-Tyr(tBu)-OH to the obtained resin in step-vi) in presence of a coupling agent and solvent to obtained protected Tirzepatide of Formula III;
viii. cleaving the protected Tirzepatide using a reagent to obtain crude Tirzepatide;
ix. purifying the crude Tirzepatide by preparative HPLC to obtain pure Tirzepatide.

4. A compound of Formula III is Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(Oxa)-Asp(OtBu)-Tyr(tBu)-Ser(Oxa)-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(AEEAc-AEEAc-?-Glu-OtBu-19-carboxynonadecanoyl mono t-butylester)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(Oxa)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Rink amide MBHA resin/ Sieber Amide resin.

5. The process as claimed in claim 1 and 4, 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.

6. The process as claimed in claim 1 and 4, 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 HCl) or a mixture thereof.

7. The process as claimed in claim 1 and 4, 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.