DESC:Title of the Invention
An improved process for the preparation of Tirzepatide and its Fragments.

Field of the Invention
The present invention relates to an improved process for the preparation of Tirzepatide having the chemical structural Formula I.

The present invention also relates to novel Fragments-2, 3, 4, 5 and its process of preparation, which are useful in the preparation of Tirzepatide.

The present invention also relates to novel compounds of Formulae II, III, IIIa, V, Va 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(X)-Asp(OtBu)-Tyr(tBu)-Ser(X)-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(Linker)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(X)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2.

Formula III: H-Thr(tBu)-Phe-Thr(tBu)-Ser(X)-Asp(OtBu)-Tyr(tBu)-Ser(X)-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(Linker)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(X)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2.

Formula IIIa: Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(X)-Asp(OtBu)-Tyr(tBu)-Ser(X)-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(Linker)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(X)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2.

Formula V: H-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(X)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2.

Formula Va: Fmoc-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(X)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2.

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 diabetes3 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.

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 hybrid approach.

The present invention provides a cost effective, novel and an efficient process for the preparation of Tirzepatide and intermediates by making appropriate fragments in a solid phase approach followed by condensing these fragments using solution phase approach with higher yields and purity.

In one embodiment, the present invention relates to an improved process for the preparation of Tirzepatide by using six fragments through hybrid approach. This process will involve the coupling of appropriate fragments which are synthesised on solid support in a required sequence, deprotection and condensing them in solution phase, followed by purification on reverse phase HPLC, freeze drying and isolation to get pure Tirzepatide.

The present invention provides a hybrid approach for the preparation of Tirzepatide compound of Formula I.

which comprises:
i) synthesis of Fragments-1, -2, -3, -4, 5 and -6 on solid support;
ii) Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-OH (Fragment 1);
iii) Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(X)-Asp(OtBu)-Tyr(tBu)-Ser(X)-Ile-Aib-OH (Fragment 2), where in X is (tBu) or (Oxa);
iv) Fmoc-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(Linker)-OH (Fragment 3);
v) Fmoc-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-OH (Fragment 4);
vi) Fmoc-Gly-Pro-Ser(tBu)-Ser(X)-Gly-OH (Fragment 5), where in X is (tBu) or (Oxa);
vii) H-Ala-Pro-Pro-Pro-Ser(tBu)-NH2 (Fragment 6)
viii) condensing Fmoc-Gly-Pro-Ser(tBu)-Ser(X)-Gly-OH (Fragment 5) with H-Ala-Pro-Pro-Pro-Ser(tBu)-NH2 (Fragment 6) in presence of coupling agent and solvent in in-situ manner followed by deprotection in presence of base to obtain H-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2 compound of Formula VI;
ix) condensing H-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2 compound of Formula VI with Fmoc-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-OH (Fragment 4) in presence of coupling agent and solvent in in-situ manner followed by deprotection in presence of base to obtain H-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2 compound of Formula V;
x) condensing H-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2 compound of Formula V with Fmoc-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(Linker)-OH (Fragment 3) in presence of coupling agent and solvent in in-situ manner followed by deprotection in presence of base to obtain H-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(Linker)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2 compound of Formula IV;
xi) condensing H-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(Linker)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2 compound of Formula IV with Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(X)-Asp(OtBu)-Tyr(tBu)-Ser(X)-Ile-Aib-OH (Fragment 2) in presence of coupling agent and solvent in in-situ manner followed by deprotection in presence of base to obtain H-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(Linker)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2 compound of Formula III;
xii) condensing H-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(Linker)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2 compound of Formula III with Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-OH (Fragment 1) in presence of coupling agent and solvent to obtain protected Tirzepatide;
xiii) cleaving the protected Tirzepatide using a reagent to obtain crude Tirzepatide;
xiv) purifying the crude Tirzepatide by preparative HPLC to obtain pure Tirzepatide.

In another embodiment, the present invention relates to novel fragment of Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(X)-Asp(OtBu)-Tyr(tBu)-Ser(X)-Ile-Aib-OH (Fragment 2), where in X is (tBu) or (Oxa)

In another embodiment, the present invention relates to novel fragment of Fmoc-Leu-Asp(tBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(Linker)-OH (Fragment 3)

In another embodiment, the present invention relates to novel fragment of Fmoc-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-OH (Fragment 4)

In another embodiment, the present invention relates to novel fragment of Fmoc-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-OH (Fragment 5), where in X is (tBu) or (Oxa)

In another embodiment, the present invention relates to novel compound 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(Linker)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2 compound of Formula II.

In another embodiment, the present invention relates to novel compound of H-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(Linker)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2 compound of Formula III.
In another embodiment, the present invention relates to novel compound of Fmoc-T(tBu)-F-T(tBu)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(Linker)-A-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 IIIa.

In another embodiment, the present invention relates to novel compound 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 V.

In another embodiment, the present invention relates to novel compound of Fmoc-A-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 Va.

The present invention relates to a novel solid phase peptide process for the preparation of Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-OH of Fragment 1

which comprises:
i) anchoring Fmoc-Gly-OH to a resin in presence of a base;
ii) selective deprotection of amino acid using a base;
iii) coupling of Fmoc-Glu(OtBu)-OH to a resin obtained in step-ii) in presence of coupling agent in a solvent to obtain dipeptide resin;
iv) sequential coupling of Fmoc-Aib-OH and Boc-Tyr(tBu)-OH to the obtained resin in step-iii) in presence of a coupling agent;
v) cleaving of protected peptide from solid support resin in presence of a reagent to obtain 4 amino acid chain peptide of Fragment 1.
The present invention relates to a novel solid phase peptide process for the preparation of Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(X)-Asp(OtBu)-Tyr(tBu)-Ser(X)-Ile-Aib-OH of Fragment 2.

which comprises:
i) anchoring Fmoc-Aib-OH to resin in presence of a base;
ii) selective deprotection of amino acid using a base;
iii) coupling of Fmoc-Ile-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-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH and Fmoc-Thr(tBu)-OH to the obtained resin in step-iii) in presence of a coupling agent;
(Optionally) sequential deprotection and coupling of Fmoc-Tyr(tBu)-Ser(Oxa)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Thr(tBu)-Ser(Oxa)-OH, Fmoc-Phe-OH and Fmoc-Thr(tBu)-OH to the obtained resin in step-iii) in presence of a coupling agent;
v) cleaving of protected peptide from solid support resin in presence of a reagent to obtain Fragment 2.

The present invention relates to a novel solid phase peptide process for the preparation of H-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(Linker)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2 of Fragment 3.

which comprises:
i) anchoring Fmoc-Lys(Linker)-OH to resin in presence of a base;
ii) selective deprotection of amino acid using a base;
iii) coupling of Fmoc-Gln(Trt)-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-Ala-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH and Fmoc-Leu-OH to the obtained resin in step-iii) in presence of a coupling agent;
v) cleavage of protected peptide from solid support resin in presence of a reagent to obtain Fragment 3.

The present invention relates to a novel solid phase peptide process for the preparation of Fmoc-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-OH of Fragment 4.

which comprises:
i) anchoring Fmoc-Gly-OH to a resin in presence of a base;
ii) selective deprotection of amino acid using a base;
iii) coupling of Fmoc-Ala-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-Ile-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH and Fmoc-Ala-OH to the obtained resin in step-iii) in presence of a coupling agent;
v) cleavage of protected peptide from solid support resin in presence of a reagent to obtain Fragment 4.

The present invention relates to a novel solid phase peptide process for the preparation of Fmoc-Gly-Pro-Ser(tBu)-Ser(X)-Gly-OH of Fragment 5.

which comprises:
i) anchoring Fmoc-Gly-OH to a resin in presence of a base;
ii) selective deprotection of amino acid using a base;
iii) coupling of Fmoc-Ser(tBu)-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-Ser(tBu)-OH, Fmoc-Pro-OH and Fmoc-Gly-OH to the obtained resin in step-iii) in presence of a coupling agent;
(Optionally) coupling of Fmoc-Ser(tBu)-Ser(Oxa)-OH to a resin obtained in step-ii) in presence of coupling agent in a solvent to obtain dipeptide resin; sequential deprotection and coupling of Fmoc-Pro-OH and Fmoc-Gly-OH to the obtained resin in step-iii) in presence of a coupling agent;
v) cleavage of protected peptide from solid support resin in presence of a reagent to obtain Fragment 5.

The present invention relates to a novel solid phase peptide process for the preparation of NH2-Ala-Pro-Pro-Pro-Ser(tBu)-NH2 of Fragment 6.

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 and Fmoc-Ala-OH to the obtained resin in step-iii) in presence of a coupling agent;
v) cleavage of protected peptide from solid support resin in presence of a reagent to obtain Fragment 6.

Detailed Description of the Invention

The present invention provides an improved process for the preparation of Tirzepatide by making appropriate fragments on solid support, followed by condensing these fragments using solution phase approach with higher yields and purity.

The novel fragments and compounds which are used in the preparation of Tirzepatide are as follows.
Fragment 2: Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(X)-Asp(OtBu)-Tyr(tBu)-Ser(X)-Ile-Aib-OH, where in X is (tBu) or (Oxa)
Fragment 3: Fmoc-Leu-Asp(tBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(Linker)-OH.
Fragment 4: Fmoc-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-OH.
Fragment 5: Fmoc-Gly-Pro-Ser(tBu)-Ser(X)-Gly-OH, where in X is (tBu) or (Oxa)
Formula II: Boc-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(Boc)-I-A-Q(Trt)-K(Linker)-A-F-V-Q(Trt)-W(Boc)-L-I-A-G-G-P-S(tBu)-S(tBu)-G-A-P-P-P-S(tBu)-NH2.
Formula III: H-T(tBu)-F-T(tBu)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(Linker)-A-F-V-Q-(Trt)-W(Boc)-L-I-A-G-G-P-S(tBu)-S(tBu)-G-A-P-P-P-S(tBu)-NH2.
Formula IIIa: Fmoc-T(tBu)-F-T(tBu)-S(tBu)-D(OtBu)-Y(tBu)-S(tBu)-I-Aib-L-D(OtBu)-K(Boc)-I-A-Q(Trt)-K(Linker)-A-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-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2.
Formula Va: Fmoc-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2.
Linker: AEEA-AEEA-?-Glu-Eicosanedioic acid (or) AEEA-AEEA-?-Glu (a-OtBu)-Eicosanedioic acid mono-t-butyl.

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

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 chloro trityl resin (CTC), wang resin, 4-methyltrityl chloride, sieber amide resin and rink acid resin. Preferably using 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.

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, N-methyl morpholine and mixture thereof.

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 in the reaction can be selected from the group consisting of Ethylcyano (hydroxyimino)acetate-02)-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 its 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 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.

In one embodiment, the present invention relates to an improved process for the preparation of Tirzepatide by coupling appropriate fragments 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), synthesis of Fragments-1, -2, -3, -4, 5 and -6 on solid support;
Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-OH (Fragment 1)
Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(X)-Asp(OtBu)-Tyr(tBu)-Ser(X)-Ile-Aib-OH (Fragment 2), where in X is (tBu) or (Oxa)
Fmoc-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(Linker)-OH (Fragment 3)
Fmoc-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-OH (Fragment 4)
Fmoc-Gly-Pro-Ser(tBu)-Ser(X)-Gly-OH (Fragment 5), where in X is (tBu) or (Oxa)
H-Ala-Pro-Pro-Pro-Ser(tBu)-NH2 (Fragment 6)

In step ii), condensation of Fmoc-Gly-Pro-Ser(tBu)-Ser(X)-Gly-OH (Fragment 5) with
H-Ala-Pro-Pro-Pro-Ser(tBu)-NH2 (Fragment 6) in presence of a coupling agent and
suitable solvent or mixture of organic solvents in in-situ manner followed deprotection in
presence of base to obtain H-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(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 3 hours to 5 hours, preferably for a period of 4 hours.

In step iii), condensing H-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2 compound of Formula VI with Fmoc-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-OH (Fragment 4) in presence of coupling agent and solvent in in-situ manner followed by deprotection in presence of base to obtain H-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2 compound of Formula V;

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 iv), condensing H-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2 compound of Formula V with Fmoc-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(Linker)-OH (Fragment-3) in presence of coupling agent and solvent in in-situ manner followed by deprotection in presence of base to obtain H-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(Linker)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(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 3 hour to 8 hours, preferably for a period of 7 hours.

In step v), condensing H-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(Linker)-Ala-Phe-Val-Gln(Trt0-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2 compound of Formula IV with Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(X)-Asp(OtBu)-Tyr(tBu)-Ser(X)-Ile-Aib-OH (Fragment-2) in presence of coupling agent and solvent in in-situ manner followed by deprotection in presence of base to obtain H-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(Linker)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(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 3 hour to 8 hours, preferably for a period of 7 hours.

In step vi), condensing H-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(Linker)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2 compound of Formula III with Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-OH (Fragment 1) in presence of coupling agent and solvent to obtain 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 vii) cleaving the 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%/1% to 95%/10%/10%/5%, preferably cocktail mixture is 90%/5%/5%/2.5%.

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

In another embodiment, the present invention relates to a novel solid phase peptide process for the preparation of Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-OH of Fragment 1 by using solution phase synthesis, which is useful in the preparation of Tirzepatide. The process will involve the coupling of required sequence, cleavage and deprotection, followed by purification to get Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-OH of Fragment 1. which comprises:
i) anchoring Fmoc-Gly-OH to a resin in presence of a base;
ii) selective deprotection of amino acid using a base;
iii) coupling of Fmoc-Glu(OtBu)-OH to a resin obtained in step-ii) in presence of coupling agent in a solvent to obtain dipeptide resin;
iv) sequential coupling of Fmoc-Aib-OH and Boc-Tyr(tBu)-OH to the obtained resin in step-iii) in presence of a coupling agent;
v) cleaving of protected peptide from solid support resin in presence of a reagent to obtain 4 amino acid chain peptide of Fragment 1.

In step-i), 2-CTC resin was taken in a SPPS reactor and swollen by adding of dichloromethane (MDC). Fmoc-Gly-OH and diisopropylethylamine (DIEA) was added to the resulting reaction mixture in presence of dichloromethane (MDC).

In step-ii), deprotecting the Fmoc group in presence of a base, preferably using 20% piperidine in dimethylformamide (or) 1.5% DBU in dichloromethane (MDC).

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

In step-iv), sequential addition of Fmoc-Aib-OH and Boc-Tyr(tBu)-OH to obtained resin in step-i) in presence of coupling agent.
The coupling agent used in this step is DIC, Oxyma in DMF.
The reaction temperature may range from 25 °C to 30 °C. The duration of the reaction may range from 1 to 4 hours, preferably for the period of 2-3 hours.
Deprotection carried out using 20 % of piperidine in Dimethyl formamide.

In step-v), deprotection is carried out for protected peptide from solid support resin using a reagent to obtained 4 amino acid peptide of Fragment 1.

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. Preferably using TFA in dichloromethane.

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

In another embodiment, the present invention relates to a novel solid phase peptide process for the preparation of Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(X)-Asp(OtBu)-Tyr(tBu)-Ser(X)-Ile-Aib-OH (Fragment 2), by using solution phase synthesis, which is useful in the preparation of Tirzepatide. The process will involve the coupling of required sequence, cleavage and deprotection, followed by purification to get Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(X)-Asp(OtBu)-Tyr(tBu)-Ser(X)-Ile-Aib-OH (Fragment 2). which comprises:
i) anchoring Fmoc-Aib-OH to resin in presence of a base;
ii) selective deprotection of amino acid using a base;
iii) coupling of Fmoc-Ile-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-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH and Fmoc-Thr(tBu)-OH to the obtained resin in step-iii) in presence of a coupling agent;
(Optionally) sequential deprotection and coupling of Fmoc-Tyr(tBu)-Ser(Oxa)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Thr(tBu)-Ser(Oxa)-OH, Fmoc-Phe-OH and Fmoc-Thr(tBu)-OH to the obtained resin in step-iii) in presence of a coupling agent;
v) cleaving of protected peptide from solid support resin in presence of a reagent to obtain Fragment 2.

In step-i), 2-CTC resin was taken in a SPPS reactor and swollen by adding dichloromethane (MDC). The first amino acid Fmoc-Aib-OH and diisopropylethylamine (DIEA) was added to the resulting reaction mixture in presence of dichloromethane (MDC).

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 25 °C to 30 °C. The duration of the reaction may range from 1 to 4 hours, preferably for the period of 2-3 hours.

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

In step-iv), sequential cleavage and addition of Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH and Fmoc-Thr(tBu)-OH to the obtained resin in step-iii) in presence of a coupling agent.
(Optionally) sequential deprotection and coupling of Fmoc-Tyr(tBu)-Ser(Oxa)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Thr(tBu)-Ser(Oxa)-OH, Fmoc-Phe-OH and Fmoc-Thr(tBu)-OH to the obtained resin in step-iii) in presence of a coupling agent;
The coupling agent used in this step is using DIC, Oxyma pure in DMF.
The reaction temperature may range from 25 °C to 30 °C. The duration of the reaction may range from 1 to 4 hours, preferably for the period of 2-3 hours.
Deprotection carried out using 20 % of piperidine in Dimethylformamide.
In step-v), cleavage is carried out for protected peptide from solid support resin using a reagent to obtained Fragment 2.
The reaction was monitored by Kaiser test.

In another embodiment, the present invention relates to a novel solid phase peptide process for the preparation of Fmoc-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(Linker)-OH (Fragment 3) by using solution phase synthesis, which is useful in the preparation of Tirzepatide. The process will involve the coupling of required sequence, cleavage and deprotection, followed by purification to get Fmoc-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(Linker)-OH (Fragment 3). which comprises:
i) anchoring Fmoc-Lys(Linker)-OH to resin in presence of a base;
ii) selective deprotection of amino acid using a base;
iii) coupling of Fmoc-Gln(Trt)-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-Ala-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH and Fmoc-Leu-OH to the obtained resin in step-iii) in presence of a coupling agent;
v) cleavage of protected peptide from solid support resin in presence of a reagent to obtain Fragment 3.

In step-i), 2-CTC resin was taken in a SPPS reactor and swollen by adding dichloromethane (MDC). The first amino acid Fmoc-Lys(Linker)-OH and diisopropylethylamine (DIEA) was added to the resulting reaction mixture in presence of dichloromethane (MDC).

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 25 °C to 30 °C. The duration of the reaction may range from 1 to 4 hours, preferably for the period of 2-3 hours.

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

In step-iv), sequential cleavage and addition of Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH and Fmoc-Leu-OH to the obtained resin in step-iii) in presence of a coupling agent.
The coupling agent used in this step is using DIC, Oxyma pure in DMF.
The reaction temperature may range from 25 °C to 30 °C. The duration of the reaction may range from 1 to 4 hours, preferably for the period of 2-3 hours.
Deprotection carried out using 20 % of piperidine in Dimethylformamide.

In step-v), cleavage is carried out for protected peptide from solid support resin using a reagent to obtained Fragment 3.
The reaction was monitored by Kaiser test.

In another embodiment, the present invention relates to a novel solid phase peptide process for the preparation of Fmoc-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-OH (Fragment 4), by using solution phase synthesis, which is useful in the preparation of Tirzepatide. The process will involve the coupling of required sequence, cleavage and deprotection, followed by purification to get Fmoc-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-OH (Fragment 4). which comprises:
i) anchoring Fmoc-Gly-OH to a resin in presence of a base;
ii) selective deprotection of amino acid using a base;
iii) coupling of Fmoc-Ala-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-Ile-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH and Fmoc-Ala-OH to the obtained resin in step-iii) in presence of a coupling agent;
v) cleavage of protected peptide from solid support resin in presence of a reagent to obtain Fragment 4.

In step-i), 2-CTC resin was taken in a SPPS reactor and swollen by adding dichloromethane (MDC). The first amino acid Fmoc-Gly-OH and diisopropylethylamine (DIEA) was added to the resulting reaction mixture in presence of dichloromethane (MDC).

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 25 °C to 30 °C. The duration of the reaction may range from 1 to 4 hours, preferably for the period of 2-3 hours.

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

In step-iv), sequential cleavage and addition of Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH and Fmoc-Ala-OH to the obtained resin in step-iii) in presence of a coupling agent.
The coupling agent used in this step is using DIC, Oxyma pure in DMF.
The reaction temperature may range from 25 °C to 30 °C. The duration of the reaction may range from 1 to 4 hours, preferably for the period of 2-3 hours.
Deprotection carried out using 20 % of piperidine in Dimethylformamide.

In step-v), cleavage is carried out for protected peptide from solid support resin using a reagent to obtained Fragment 4.
The reaction was monitored by Kaiser test.

In another embodiment, the present invention relates to a novel solid phase peptide process for the preparation of Fmoc-Gly-Pro-Ser(tBu)-Ser(X)-Gly-OH (Fragment 5), by using solution phase synthesis, which is useful in the preparation of Tirzepatide. The process will involve the coupling of required sequence, cleavage and deprotection, followed by purification to get Fmoc-Gly-Pro-Ser(tBu)-Ser(X)-Gly-OH (Fragment 5). which comprises:
i) anchoring Fmoc-Gly-OH to a resin in presence of a base;
ii) selective deprotection of amino acid using a base;
iii) coupling of Fmoc-Ser(tBu)-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-Ser(tBu)-OH, Fmoc-Pro-OH and Fmoc-Gly-OH to the obtained resin in step-iii) in presence of a coupling agent;
(Optionally) coupling of Fmoc-Ser(tBu)-Ser(Oxa)-OH to a resin obtained in step-ii) in presence of coupling agent in a solvent to obtain dipeptide resin; sequential deprotection and coupling of Fmoc-Pro-OH and Fmoc-Gly-OH to the obtained resin in step-iii) in presence of a coupling agent;
v) cleavage of protected peptide from solid support resin in presence of a reagent to obtain Fragment 5.

In step-i), 2-CTC resin was taken in a SPPS reactor and swollen by adding dichloromethane (MDC). The first amino acid Fmoc-Gly-OH and diisopropylethylamine (DIEA) was added to the resulting reaction mixture in presence of dichloromethane (MDC).

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 25 °C to 30 °C. The duration of the reaction may range from 1 to 4 hours, preferably for the period of 2-3 hours.

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

In step-iv), sequential cleavage and addition of Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH and Fmoc-Gly-OH to the obtained resin in step-iii) in presence of a coupling agent.
(Optionally) coupling of Fmoc-Ser(tBu)-Ser(Oxa)-OH to a resin obtained in step-ii) in presence of coupling agent in a solvent to obtain dipeptide resin; sequential deprotection and coupling of Fmoc-Pro-OH and Fmoc-Gly-OH to the obtained resin in step-iii) in presence of a coupling agent;

The coupling agent used in this step is using DIC, Oxyma pure in DMF. The reaction temperature may range from 25 °C to 30 °C. The duration of the reaction may range from 1 to 4 hours, preferably for the period of 2-3 hours.

Deprotection carried out using 20 % of piperidine in Dimethylformamide.

In step-v), cleavage is carried out for protected peptide from solid support resin using a reagent to obtained Fragment 5.

The reaction was monitored by Kaiser test.

In another embodiment, the present invention relates to a novel solid phase peptide process for the preparation of NH2-Ala-Pro-Pro-Pro-Ser(tBu)-NH2 (Fragment 6), by using solution phase synthesis, which is useful in the preparation of Tirzepatide. The process will involve the coupling of required sequence, cleavage and deprotection, followed by purification to get NH2-Ala-Pro-Pro-Pro-Ser(tBu)-NH2 (Fragment 6). 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 and Fmoc-Ala-OH to the obtained resin in step-iii) in presence of a coupling agent;
v) cleavage of protected peptide from solid support resin in presence of a reagent to obtain Fragment 6.

In step-i), sieber amide resin was taken in a SPPS reactor and swollen by adding dichloromethane (MDC). The first amino acid Fmoc-Ser(tBu)-OH and diisopropylethylamine (DIEA) was added to the resulting reaction mixture in presence of dichloromethane (MDC).

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 25 °C to 30 °C. The duration of the reaction may range from 1 to 4 hours, preferably for the period of 2-3 hours.

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

In step-iv), sequential cleavage and addition of Fmoc-Pro-OH, Fmoc-Pro-OH and Fmoc-Ala-OH to the obtained resin in step-iii) in presence of a coupling agent.

The coupling agent used in this step is using DIC, Oxyma pure in DMF. The reaction temperature may range from 25 °C to 30 °C. The duration of the reaction may range from 1 to 4 hours, preferably for the period of 2-3 hours.

Deprotection carried out using 20 % of piperidine in Dimethylformamide.
In step-v), cleavage is carried out for protected peptide from solid support resin using a reagent to obtained Fragment 6.
The reaction was monitored by 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: 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.

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.
Preparation-1: Solid phase peptide synthesis of Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-OH (Fragment 1)
Step-A: 2-CTC resin (40 grams) was taken in a SPPS reactor and dichloromethane (20 mL) was added and allowed it to swell for 10 minutes.

Step-B: A solution of Fmoc-Gly-OH (38.0 grams) and Diisopropylethylamine (69.67 mL) in dry dichloromethane (320 mL) was added to the resin obtained from step-A and stirred for 2 hours at room temperature. The above resin was deblocked with 20 % piperidine in DMF (200 mL) for 10-15 minutes and washed with DMF.

Step-C: Fmoc-Glu(OtBu)-OH (51.3 grams) was dissolved in DMF (320 mL) and stirred for 10 minutes. DIC (18.6 mL) and oxyma (17.1 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with DMF (320 mL), isopropanol (320 mL) and dichloromethane (320 mL). The resulting resin was deblocked with 20 % piperidine in DMF (200 mL) for 10 minutes and washed with DMF (320 mL).

Step-D: Fmoc-Aib-OH (39.0 grams) was dissolved in DMF (320 mL) and stirred for 10 minutes. DIC (18.6 mL) and oxyma (17.1 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with DMF (320 mL), isopropanol (320 mL) and dichloromethane (320 mL). The resulting resin was deblocked with 20 % piperidine in DMF (200 mL) for 10 minutes and washed with DMF (320 mL).

Step-E: Boc-Tyr(tBu)-OH (26.9 grams) was dissolved in DMF (320 mL) and stirred for 10 minutes. DIC (18.6 mL) and oxyma (17.1 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was drained and washed with DMF (320 mL), isopropanol (320 mL) and dichloromethane (320 mL). The resulting resin was deblocked with 20 % piperidine in DMF (200 mL).

Step-G: 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 MeOH (400 mL).

Step-H: Selective cleavage of CTC-resin from Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-CTC resin was performed with a mixture of Trifluoroacetic acid (8 mL) in dichloromethane (792 mL). The crude protected peptide was isolated by precipitating with ether.

Yield: 45.0 grams.
Purity: 95.8% (HPLC purity)

Preparation-2:
Method-1: Solid phase peptide synthesis of Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-OH (Fragment 2)

Step-A: 2-CTC resin (50 grams) was taken in a SPPS reactor and dichloromethane (120 mL) was added and allowed it to swell for 10 minutes.

Step-B: A solution of Fmoc-Aib-OH (52.0 grams) and Diisopropylethylamine (69.8 mL) in dry dichloromethane (400 mL) was added to the resin obtained from step-A and stirred for 2 hours at room temperature. The above resin was deblocked with 20 % piperidine in DMF (200 mL) for 10-15 minutes and washed with DMF.

Step-C: Fmoc-Ile-OH (47.17 grams) was dissolved in DMF (400 mL) and stirred for 10 minutes. DIC (20.6 mL) and oxyma (18.9 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with DMF (400 mL), isopropanol (400 mL) and dichloromethane (400 mL). The resulting resin was deblocked with 20 % piperidine in DMF (200 mL) for 10 minutes and washed with DMF (400 mL).

Step-D: Fmoc-Ser(tBu)-OH (51.2 grams) was dissolved in DMF (400 mL) and stirred for 10 minutes. DIC (20.6 mL) and oxyma (18.9 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with DMF (400 mL), isopropanol (400 mL) and dichloromethane (400 mL). The resulting resin was deblocked with 20 % piperidine in DMF (200 mL) for 10 minutes and washed with DMF (400 mL).

Step-E: Fmoc-Tyr(tBu)-OH (61.4 grams) was dissolved in DMF (400 mL) and stirred for 10 minutes. DIC (20.6 mL) and oxyma (18.9 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was drained and washed with DMF (400 mL), isopropanol (400 mL) and dichloromethane (400 mL). The resulting resin was deblocked with 20 % piperidine in DMF (200 mL).

Step-F: Fmoc-Asp(OtBu)-OH (55.0 grams) was dissolved in DMF (400 mL) and stirred for 10 minutes. DIC (20.6 mL) and oxyma (18.9 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was drained and washed with DMF (400 mL), isopropanol (400 mL) and dichloromethane (400 mL). The resulting resin was deblocked with 20 % piperidine in DMF (200 mL).

Step-G: Fmoc-Ser(tBu)-OH (51.18 grams) was dissolved in DMF (400 mL) and stirred for 10 minutes. DIC (20.6 mL) and oxyma (18.9 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was drained and washed with DMF (400 mL), isopropanol (400 mL) and dichloromethane (400 mL). The resulting resin was deblocked with 20 % piperidine in DMF (200 mL).

Step-H: Fmoc-Asp(OtBu)-OH (18.49 grams) was dissolved in DMF (400 mL) and stirred for 10 minutes. DIC (20.6 mL) and oxyma (18.9 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was drained and washed with DMF (400 mL), isopropanol (400 mL) and dichloromethane (400 mL). The resulting resin was deblocked with 20 % piperidine in DMF (200 mL).

Step-I: Fmoc-Thr(tBu)-OH (53.0 grams) was dissolved in DMF (400 mL) and stirred for 10 minutes. DIC (20.6 mL) and oxyma (18.9 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was drained and washed with DMF (400 mL), isopropanol (400 mL) and dichloromethane (400 mL). The resulting resin was deblocked with 20 % piperidine in DMF (200 mL).

Step-J: Fmoc-Phe-OH (51.8 grams) was dissolved in DMF (400 mL) and stirred for 10 minutes. DIC (20.6 mL) and oxyma (18.9 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was drained and washed with DMF (400 mL), isopropanol (400 mL) and dichloromethane (400 mL). The resulting resin was deblocked with 20 % piperidine in DMF (200 mL).

Step-K: Fmoc-Thr(tBu)-OH (53.0 grams) was dissolved in DMF (400 mL) and stirred for 10 minutes. DIC (20.6 mL) and oxyma (18.9 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was drained and washed with DMF (400 mL), isopropanol (400 mL) and dichloromethane (400 mL). The resulting resin was deblocked with 20 % piperidine in DMF (200 mL).

Step-L: Coupling of the last amino acid the resin was washed with DMF (400 mL), Isopropanol (200 mL) and MDC (200 mL) then dried and washed with MeOH (500 mL).

Step-M: Selective cleavage of CTC-resin from Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-CTC resin was performed with a mixture of Trifluoroacetic acid (16 mL) in dichloromethane (1584 mL). The crude protected peptide was isolated by precipitating with n-heptane.

Yield: 34.0 grams.
Purity: 89.0% (HPLC purity)

Method-2: Solid phase peptide synthesis of Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(Oxa)-Asp(OtBu)-Tyr(tBu)-Ser(Oxa)-Ile-Aib-OH (Fragment 2)

Step-A: 2-CTC resin (50 grams) was taken in a SPPS reactor and dichloromethane (120 mL) was added and allowed it to swell for 10 minutes.

Step-B: A solution of Fmoc-Aib-OH (52.0 grams) and Diisopropylethylamine (69.8 mL) in dry dichloromethane (400 mL) was added to the resin obtained from step-A and stirred for 2 hours at room temperature. The above resin was deblocked with 20 % piperidine in DMF (200 mL) for 10-15 minutes and washed with DMF.

Step-C: Fmoc-Ile-OH (47.17 grams) was dissolved in DMF (400 mL) and stirred for 10 minutes. DIC (20.6 mL) and oxyma (18.9 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with DMF (400 mL), isopropanol (400 mL) and dichloromethane (400 mL). The resulting resin was deblocked with 20 % piperidine in DMF (200 mL) for 10 minutes and washed with DMF (400 mL).

Step-D: Fmoc-Tyr(tBu)-Ser(Oxa)-OH (51.2 grams) was dissolved in DMF (400 mL) and stirred for 10 minutes. DIC (20.6 mL) and oxyma (18.9 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with DMF (400 mL), isopropanol (400 mL) and dichloromethane (400 mL). The resulting resin was deblocked with 20 % piperidine in DMF (200 mL) for 10 minutes and washed with DMF (400 mL).

Step-E: Fmoc-Asp(OtBu)-OH (55.0 grams) was dissolved in DMF (400 mL) and stirred for 10 minutes. DIC (20.6 mL) and oxyma (18.9 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was drained and washed with DMF (400 mL), isopropanol (400 mL) and dichloromethane (400 mL). The resulting resin was deblocked with 20 % piperidine in DMF (200 mL).
Step-F: Fmoc-Ser(tBu)-OH (51.18 grams) was dissolved in DMF (400 mL) and stirred for 10 minutes. DIC (20.6 mL) and oxyma (18.9 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was drained and washed with DMF (400 mL), isopropanol (400 mL) and dichloromethane (400 mL). The resulting resin was deblocked with 20 % piperidine in DMF (200 mL).

Step-G: Fmoc-Asp(OtBu)-OH (18.49 grams) was dissolved in DMF (400 mL) and stirred for 10 minutes. DIC (20.6 mL) and oxyma (18.9 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was drained and washed with DMF (400 mL), isopropanol (400 mL) and dichloromethane (400 mL). The resulting resin was deblocked with 20 % piperidine in DMF (200 mL).

Step-H: Fmoc-Thr(tBu)-OH (53.0 grams) was dissolved in DMF (400 mL) and stirred for 10 minutes. DIC (20.6 mL) and oxyma (18.9 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was drained and washed with DMF (400 mL), isopropanol (400 mL) and dichloromethane (400 mL). The resulting resin was deblocked with 20 % piperidine in DMF (200 mL).

Step-I: Fmoc-Phe-OH (51.8 grams) was dissolved in DMF (400 mL) and stirred for 10 minutes. DIC (20.6 mL) and oxyma (18.9 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was drained and washed with DMF (400 mL), isopropanol (400 mL) and dichloromethane (400 mL). The resulting resin was deblocked with 20 % piperidine in DMF (200 mL).

Step-J: Fmoc-Thr(tBu)-OH (53.0 grams) was dissolved in DMF (400 mL) and stirred for 10 minutes. DIC (20.6 mL) and oxyma (18.9 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was drained and washed with DMF (400 mL), isopropanol (400 mL) and dichloromethane (400 mL). The resulting resin was deblocked with 20 % piperidine in DMF (200 mL).

Step-K: Coupling of the last amino acid the resin was washed with DMF (400 mL), Isopropanol (200 mL) and MDC (200 mL) then dried and washed with MeOH (500 mL).

Step-L: Selective cleavage of CTC-resin from Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-CTC resin was performed with a mixture of Trifluoroacetic acid (16 mL) in dichloromethane (1584 mL). The crude protected peptide was isolated by precipitating with n-heptane.

Yield: 35.0 grams
Purity: 91.0% (HPLC purity)

Preparation-3: Solid phase peptide synthesis of Fmoc-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(Linker)-OH (Fragment 3)

Step-A: 2-CTC resin (30 grams) was taken in a SPPS reactor and dichloromethane (120 mL) was added and allowed it to swell for 10 minutes.

Step-B: A solution of Fmoc-Lys(Linker)-OH (58.7 grams) and Diisopropylethylamine (41.8 mL) in dry dichloromethane (240 mL) was added to the resin obtained from step-A and stirred for 2 hours at room temperature. The above resin was deblocked with 20 % piperidine in DMF (50 mL) for 10-15 minutes and washed with DMF.

Step-C: Fmoc-Gln(Trt)-OH (27.4 grams) was dissolved in DMF (240 mL) and stirred for 10 minutes. DIC (6.96 mL) and oxyma (6.39 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with DMF (240 mL), isopropanol (240 mL) and dichloromethane (240 mL). The resulting resin was deblocked with 20 % piperidine in DMF (150 mL) for 10 minutes and washed with DMF (240 mL).

Step-D: Fmoc-Ala-OH (14.0 grams) was dissolved in DMF (240 mL) and stirred for 10 minutes. DIC (6.96 mL) and oxyma (6.39 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with DMF (240 mL), isopropanol (240 mL) and dichloromethane (240 mL). The resulting resin was deblocked with 20 % piperidine in DMF (150 mL) for 10 minutes and washed with DMF (240 mL).

Step-E: Fmoc-Ile-OH (15.9 grams) was dissolved in DMF (240 mL) and stirred for 10 minutes. DIC (6.96 mL) and oxyma (6.39 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was drained and washed with DMF (240 mL), isopropanol (240 mL) and dichloromethane (200 mL). The resulting resin was deblocked with 20 % piperidine in DMF (150 mL).

Step-F: Fmoc-Lys(Boc)-OH (21.0 grams) was dissolved in DMF (240 mL) and stirred for 10 minutes. DIC (6.96 mL) and oxyma (6.39 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was drained and washed with DMF (240 mL), isopropanol (240 mL) and dichloromethane (240 mL). The resulting resin was deblocked with 20 % piperidine in DMF (150 mL).

Step-G: Fmoc-Asp(OtBu)-OH (18.5 grams) was dissolved in DMF (240 mL) and stirred for 10 minutes. DIC (6.96 mL) and oxyma (6.39 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was drained and washed with DMF (240 mL), isopropanol (240 mL) and dichloromethane (240 mL). The resulting resin was deblocked with 20 % piperidine in DMF (150 mL).

Step-H: Fmoc-Leu-OH (15.9 grams) was dissolved in DMF (240 mL) and stirred for 10 minutes. DIC (6.96 mL) and oxyma (6.39 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was drained and washed with DMF (240 mL), isopropanol (240 mL) and dichloromethane (240 mL). The resulting resin was deblocked with 20 % piperidine in DMF (150 mL).

Step-I: Coupling of the last amino acid the resin was washed with DMF (300 mLx2), Isopropanol (300 mLx2) and MDC (300 mLx2) then dried and washed with MeOH (300 mL).
x

Step-J: Selective cleavage of CTC-resin from Fmoc-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(Linker)-CTC resin was performed with a mixture of Trifluoroacetic acid (9.6 mL) in dichloromethane (950.4 mL). The crude protected peptide was isolated by precipitating with ether.

Yield: 31.0 grams.
Purity: 91.0% (HPLC purity)

Preparation-4: Solid phase peptide synthesis of Fmoc-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-OH (Fragment 4)

Step-A: 2-CTC resin (50 grams) was taken in a SPPS reactor and dichloromethane (400 mL) was added and allowed it to swell for 10 minutes.

Step-B: A solution of Fmoc-Gly-OH (47.5 grams) and Diisopropylethylamine (69.8 mL) in dry dichloromethane (400 mL) was added to the resin obtained from step-A and stirred for 2 hours at room temperature. The above resin was deblocked with 20 % piperidine in DMF (200 mL) for 10-15 minutes and washed with DMF.

Step-C: Fmoc-Ala-OH (33.64 grams) was dissolved in DMF (400 mL) and stirred for 10 minutes. DIC (16.7 mL) and oxyma (15.3 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with DMF (400 mL), isopropanol (400 mL) and dichloromethane (400 mL). The resulting resin was deblocked with 20 % piperidine in DMF (200 mL) for 10 minutes and washed with DMF (400 mL).

Step-D: Fmoc-Ile-OH (38.2 grams) was dissolved in DMF (400 mL) and stirred for 10 minutes. DIC (16.7 mL) and oxyma (15.3 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with DMF (400 mL), isopropanol (400 mL) and dichloromethane (400 mL). The resulting resin was deblocked with 20 % piperidine in DMF (200 mL) for 10 minutes and washed with DMF (400 mL).

Step-E: Fmoc-Leu-OH (38.2 grams) was dissolved in DMF (400 mL) and stirred for 10 minutes. DIC (16.7 mL) and oxyma (15.3 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was drained and washed with DMF (400 mL), isopropanol (400 mL) and dichloromethane (400 mL). The resulting resin was deblocked with 20 % piperidine in DMF (200 mL).

Step-F: Fmoc-Trp(Boc)-OH (56.87 grams) was dissolved in DMF (400 mL) and stirred for 10 minutes. DIC (16.7 mL) and oxyma (15.3 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was drained and washed with DMF (400 mL), isopropanol (400 mL) and dichloromethane (400 mL). The resulting resin was deblocked with 20 % piperidine in DMF (200 mL).

Step-G: Fmoc-Gln(Trt)-OH (65.9 grams) was dissolved in DMF (400 mL) and stirred for 10 minutes. DIC (16.7 mL) and oxyma (15.3 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was drained and washed with DMF (400 mL), isopropanol (400 mL) and dichloromethane (400 mL). The resulting resin was deblocked with 20 % piperidine in DMF (200 mL).

Step-H: Fmoc-Val-OH (36.6 grams) was dissolved in DMF (400 mL) and stirred for 10 minutes. DIC (16.7 mL) and oxyma (15.3 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was drained and washed with DMF (400 mL), isopropanol (400 mL) and dichloromethane (400 mL). The resulting resin was deblocked with 20 % piperidine in DMF (200 mL).

Step-I: Fmoc-Phe-OH (41.8 grams) was dissolved in DMF (400 mL) and stirred for 10 minutes. DIC (16.7 mL) and oxyma (15.3 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was drained and washed with DMF (400 mL), isopropanol (400 mL) and dichloromethane (400 mL). The resulting resin was deblocked with 20 % piperidine in DMF (200 mL).

Step-J: Fmoc-Ala-OH (33.6 grams) was dissolved in DMF (400 mL) and stirred for 10 minutes. DIC (16.7 mL) and oxyma (15.3 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was drained and washed with DMF (400 mL), isopropanol (400 mL) and dichloromethane (400 mL). The resulting resin was deblocked with 20 % piperidine in DMF (200 mL).

Step-K: Coupling of the last amino acid the resin was washed with DMF (500 mLx3), Isopropanol (500 mL) and MDC (500 mL) then dried and washed with MeOH (500 mL).
x

Step-L: Selective cleavage of CTC-resin from Fmoc-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-CTC resin was performed with a mixture of Trifluoroacetic acid (20 mL) in dichloromethane (1980 mL). The crude protected peptide was isolated by precipitating with ether.

Yield: 65.0 grams.
Purity: 89.1% (HPLC purity)

Preparation-5:
Method 1: Solid phase peptide synthesis of Fmoc-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-OH (Fragment 5)
Step-A: CTC resin (50 grams) was taken in a SPPS reactor and dichloromethane (20 mL) was added and allowed it to swell for 10 minutes.

Step-B: A solution of Fmoc-Gly-OH (47.5 grams) and Diisopropylethylamine (69.8 mL) in dry dichloromethane (400 mL) was added to the resin obtained from step-A and stirred for 2 hours at room temperature. The above resin was deblocked with 20 % piperidine in DMF (200 mL) for 10-15 minutes and washed with DMF.

Step-C: Fmoc-Ser(tBu)-OH (9.6 grams) was dissolved in DMF (400 mL) and stirred for 10 minutes. DIC (16.7 grams) and oxyma (15.3 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with DMF (400 mL), isopropanol (400 mL) and dichloromethane (400 mL). The resulting resin was deblocked with 20 % piperidine in DMF (200 mL) for 10 minutes and washed with DMF (400 mL).

Step-D: Fmoc-Ser(tBu)-OH (9.6 grams) was dissolved in DMF (400 mL) and stirred for 10 minutes. DIC (16.7 grams) and oxyma (15.3 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with DMF (400 mL), isopropanol (400 mL) and dichloromethane (400 mL). The resulting resin was deblocked with 20 % piperidine in DMF (400 mL) for 10 minutes and washed with DMF (400 mL).

Step-E: Fmoc-Pro-OH (8.4 grams) was dissolved in DMF (400 mL) and stirred for 10 minutes. DIC (16.7 grams) and oxyma (15.3 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was drained and washed with DMF (400 mL), isopropanol (400 mL) and dichloromethane (400 mL). The resulting resin was deblocked with 20 % piperidine in DMF (200 mL).

Step-F: Fmoc-Gly-OH (22.3 grams) was dissolved in DMF (400 mL) and stirred for 10 minutes. DIC (16.7 grams) and oxyma (15.3 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was washed with DMF (400 mL), isopropanol (400 mL) and dichloromethane.

Step-G: Coupling of the last amino acid the resin was washed with DMF (500 mLx2), Isopropanol (500 mL) and MDC (500 mL) then dried and washed with MeOH (500 mL).

Step-H: Selective cleavage of CTC-resin from Fmoc-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-CTC resin was performed with a mixture of Trifluoroacetic acid (8 mL) in dichloromethane (792 mL). The crude protected peptide was isolated by precipitating with ether.

Method 2: Solid phase peptide synthesis of Fmoc-Gly-Pro-Ser(tBu)-Ser(Oxa)-Gly-OH (Fragment 5)

Step-A: CTC resin (50 grams) was taken in a SPPS reactor and dichloromethane (20 mL) was added and allowed it to swell for 10 minutes.

Step-B: A solution of Fmoc-Gly-OH (47.5 grams) and Diisopropylethylamine (69.8 mL) in dry dichloromethane (400 mL) was added to the resin obtained from step-A and stirred for 2 hours at room temperature. The above resin was deblocked with 20 % piperidine in DMF (200 mL) for 10-15 minutes and washed with DMF.

Step-C: Fmoc-Ser(tBu)-Ser(Oxa) (9.6 grams) was dissolved in DMF (400 mL) and stirred for 10 minutes. DIC (16.7 grams) and oxyma (15.3 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with DMF (400 mL), isopropanol (400 mL) and dichloromethane (400 mL). The resulting resin was deblocked with 20 % piperidine in DMF (200 mL) for 10 minutes and washed with DMF (400 mL).

Step-D: Fmoc-Pro-OH (8.4 grams) was dissolved in DMF (400 mL) and stirred for 10 minutes. DIC (16.7 grams) and oxyma (15.3 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was drained and washed with DMF (400 mL), isopropanol (400 mL) and dichloromethane (400 mL). The resulting resin was deblocked with 20 % piperidine in DMF (200 mL).

Step-E: Fmoc-Gly-OH (22.3 grams) was dissolved in DMF (400 mL) and stirred for 10 minutes. DIC (16.7 grams) and oxyma (15.3 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was washed with DMF (400 mL), isopropanol (400 mL) and dichloromethane.

Step-F: Coupling of the last amino acid the resin was washed with DMF (500 mLx2), Isopropanol (500 mL) and MDC (500 mL) then dried and washed with MeOH (500 mL).

Step-G: Selective cleavage of CTC-resin from Fmoc-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-CTC resin was performed with a mixture of Trifluoroacetic acid (8 mL) in dichloromethane (792 mL). The crude protected peptide was isolated by precipitating with ether.

Preparation-6: Solid phase peptide synthesis of NH2-Ala-Pro-Pro-Pro-Ser(tBu)-NH2 (Fragment 6)

Step-A: Sieber amide resin (50 grams) was taken in a SPPS reactor and dichloromethane (20 mL) was added and allowed it to swell for 10 minutes.

Step-B: A solution of Fmoc-Ser(tBu)-OH (61.36 grams) and Diisopropylethylamine (69.8 mL) in dry dichloromethane (400 mL) was added to the resin obtained from step-A and stirred for 2 hours at room temperature. The above resin was deblocked with 20 % piperidine in DMF (200 mL) for 10-15 minutes and washed with DMF.

Step-C: Fmoc-Pro-OH (40.48 grams) was dissolved in DMF (400 mL) and stirred for 10 minutes. DIC (18.58 grams) and oxyma (21.3 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with DMF (400 mL), isopropanol (400 mL) and dichloromethane (400 mL). The resulting resin was deblocked with 20 % piperidine in DMF (200 mL) for 10 minutes and washed with DMF (400 mL).

Step-D: Fmoc-Pro-OH (40.48 grams) was dissolved in DMF (400 mL) and stirred for 10 minutes. DIC (18.5 grams) and oxyma (21.3 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with DMF (400 mL), isopropanol (400 mL) and dichloromethane (400 mL). The resulting resin was deblocked with 20 % piperidine in DMF (200 mL) for 10 minutes and washed with DMF (400 mL).

Step-F: Fmoc-Pro-OH (40.4 grams) was dissolved in DMF (400 mL) and stirred for 10 minutes. DIC (18.5 grams) and oxyma (21.3 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was drained and washed with DMF (400 mL), isopropanol (400 mL) and dichloromethane (400 mL). The resulting resin was deblocked with 20 % piperidine in DMF (200 mL).

Step-G: Fmoc-Gly-OH (22.3 grams) was dissolved in DMF (400 mL) and stirred for 10 minutes. DIC (16.7 grams) and oxyma (15.3 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was washed with DMF (400 mL), isopropanol (400 mL) and dichloromethane.

Step-H: Coupling of the last amino acid the resin was washed with DMF (400 mL), Isopropanol (200 mL) and MDC (200 mL) then dried and washed with MeOH (500 mL).

Step-I: Selective cleavage of sieber amide resin from NH2-Ala-Pro-Pro-Pro-Ser(tBu)-NH2-sieber amide resin was performed with a mixture of Trifluoroacetic acid (24 mL) in dichloromethane (1576 mL). The crude protected peptide was isolated by precipitating with ether.

Example-1: Process for the preparation of Tirzepatide by using six fragments through hybrid approach.

Stage-1: Process for the preparation of H-Gly-Pro-Ser(tBu)-Ser(X)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2

Fmoc-Gly-Pro-Ser(tBu)-Ser(X)-Gly-OH (10 grams) (Fragment 5) was dissolved in DMF (100 mL) and stirred for 10 minutes at 25-30 °C. H-Ala-Pro-Pro-Pro-Ser(tBu)-NH2 (8.5 grams) (Fragment 6), EDC.HCl (3.9 grams) and HOAT (3.7 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 (4.3 mL), n-heptane in DMF. Filtered the precipitated solid and washed with water and hexane to get H-Gly-Pro-Ser(tBu)-Ser(X)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2.
Yield: 10.5 grams.

Stage-2: Process for the preparation of H-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(X)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2

Fmoc-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-OH (14.0 grams) (Fragment 4) was dissolved in DMF (112 mL) then coupled with H-Gly-Pro-Ser(tBu)-Ser(X)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2 (10.0 grams) in presence of EDC.HCl (1.83 grams), HOAt (1.7 grams) in DMF (28.0 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 (9.3 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-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(X)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2.
Yield: 16.8 grams

Stage-3: Process for the preparation of H-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(Linker)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(X)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2

Fmoc-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(Linker)-OH (13.16 grams) (Fragment 3) was dissolved in DMF (375 mL) then coupled with H-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(X)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2 (15.0 grams) in presence of EDC.HCl (6.1 grams), HOAt (5.2 grams) in DMF (75.0 mL) and stirred for 10-15 minutes at 5-10 °C, maintain for 25-30 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 (12.1 mL), n-heptane (75 mL) in DMF. Filtered the precipitated solid and washed with water, hexane and DIPE to get H-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(Linker)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2.
Yield: 21.0 grams

Stage-4: Process for the preparation of H-Thr(tBu)-Phe-Thr(tBu)-Ser(X)-Asp(OtBu)-Tyr(tBu)-Ser(X)-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(Linker)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(X)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2

Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(X)-Asp(OtBu)-Tyr(tBu)-Ser(X)-Ile-Aib-OH (8.2 grams) (Fragment 2) was dissolved in DMF (525 mL) then coupled with H-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(Linker)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2 (21.0 grams) in presence of EDC.HCl (4.5 mL), HOAt (3.8 grams) in DMF (105 mL) and 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 (7.5 mL), n-heptane (105 mL) in DMF. Filtered the precipitated solid and washed with water, hexane and methanol to get H-Thr(tBu)-Phe-Thr(tBu)-Ser(X)-Asp(OtBu)-Tyr(tBu)-Ser(X)-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(Linker)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(X)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2.
Yield: 20.8 grams

Stage-5: Process for the preparation of protected Tirzepatide

Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-OH (3.4 grams) (Fragment 1) was dissolved in DMF (500 mL) then coupled with H-Thr(tBu)-Phe-Thr(tBu)-Ser(X)-Asp(OtBu)-Tyr(tBu)-Ser(X)-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(Linker)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(X)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2 (20.0 grams) in presence of EDC.HCl (3.3 grams), HOAt (2.8 grams) in DMF (100 mL) and stirred for 15-20 minutes at 5-10 °C, maintain for 4-7 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 (1.5 mL), n-heptane in DMF. Filtered the precipitated solid and washed with water and DIPE to get protected Tirzepatide.

Yield: 16.6 grams

Stage-6: Process for the preparation of crude Tirzepatide

Protected Tirzepatide was cleaved with a cocktail mixture of TFA, TIPS, water and DTT (90%/5%/5%/2.5%) in presence of DCM at 10-15 °C and stirred for 3-6 hours at the same temperature. Chilled DIPE was added to the resulting mixture and stirred for 2 hours. The precipitated solid was filtered and washed with DCM followed by DIPE to get crude Tirzepatide.

Yield: 9.0 grams

Stage-7: Preparative HPLC purification of 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.
,CLAIMS:1. An improved process for the preparation of Tirzepatide of Formula I, using hybrid approach;

which comprises:
i) condensing Fmoc-Gly-Pro-Ser(tBu)-Ser(X)-Gly-OH (Fragment 5) with H-Ala-Pro-Pro-Pro-Ser(tBu)-NH2 (Fragment 6) in presence of coupling agent and solvent in in-situ manner followed by deprotection in presence of base to obtain H-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2 compound of Formula VI;
ii) condensing H-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2 compound of Formula VI with Fmoc-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-OH (Fragment 4) in presence of coupling agent and solvent in in-situ manner followed by deprotection in presence of base to obtain H-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2 compound of Formula V;
iii) condensing H-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2 compound of Formula V with Fmoc-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(Linker)-OH (Fragment 3) in presence of coupling agent and solvent in in-situ manner followed by deprotection in presence of base to obtain H-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(Linker)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2 compound of Formula IV;
iv) condensing H-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(Linker)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2 compound of Formula IV with Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(X)-Asp(OtBu)-Tyr(tBu)-Ser(X)-Ile-Aib-OH (Fragment 2) in presence of coupling agent and solvent in in-situ manner followed by deprotection in presence of base to obtain H-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(Linker)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2 compound of Formula III;
v) condensing H-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(Linker)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2 compound of Formula III with Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-OH (Fragment 1) in presence of coupling agent and solvent to obtain protected Tirzepatide;
vi) cleaving the protected Tirzepatide using a reagent to obtain crude Tirzepatide;
vii) purifying the crude Tirzepatide by preparative HPLC to obtain pure Tirzepatide.

2. The fragments which are used in the preparation of Tirzepatide
Fragment 2: Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(X)-Asp(OtBu)-Tyr(tBu)-Ser(X)-Ile-Aib-OH, where in X is (tBu) or (Oxa);
Fragment 3: Fmoc-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(Linker)-OH;
Fragment 4: Fmoc-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-OH
Fragment 5: Fmoc-Gly-Pro-Ser(tBu)-Ser(X)-Gly-OH (Fragment 5), where in X is (tBu) or (Oxa).

3. A compound of 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(Linker)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2.
4. A compound of Formula III: H-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(Linker)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2.

5. A compound of Formula IIIa: Fmoc-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(Linker)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2.

6. A compound of Formula V: H-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2.

7. A compound of Formula Va: Fmoc-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2.

8. The process as claimed in claim 1, wherein said base is selected from group consisting of potassium carbonate, lithium carbonate, sodium carbonate, sodium hydroxide, potassium hydr 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.

9. The process as claimed in claim 1, 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.

10. The process as claimed in claim 1, 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.