DESC:FIELD OF THE INVENTION
The present invention relates to a process for the preparation of Tirzepatide, which comprises synthesis of two or more fragments and coupling the fragments to provide Tirzepatide.

BACKGROUND OF THE INVENTION
Tirzepatide, a dual agonist of Glucagon-like Peptide-1 (GLP-1) and the glucose-dependent insulinotropic polypeptide (GIP) receptors. is a 39 amino acid peptide with the following chemical name: L-tyrosyl-2-methylalanyl-L-a-glutamylglycyl-L-threonyl-Lphenylalanyl-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-Lalanylglycylglycyl-L-prolyl-L-seryl-L-serylglycyl-L-alanyl-L-prolyl-L-prolyl-L-prolyl-L-Serinamide. The amino acid sequence and structure for Tirzepatide is shown below in Formula I.

Tirzepatide is approved in the United States and other countries under the trade name MOUNJARO® and ZEPBOUND®, indicated to improve glycemic control in adults with type 2 diabetes mellitus and weight management respectively.

Tirzepatide is described in US patent No. US 9,474,780. US ‘780 patent discloses the process to prepare Tirzepatide, which involves synthesis of linear backbone by elongation of amino acid chain by solid phase synthesis (SPPS) using RAPP Rink Amide resin, followed by deprotecting the Lys20 side chain and acylating the Lys20 side chain by additional 3 coupling/deprotection cycles using a Fmoc/t-Bu strategy to obtain the protected Tirzepatide intermediate linked to a resin and then performing the concomitant cleavage from the resin and removal of side chain protecting groups to give Tirzepatide.

An international patent application WO 2020/159949 discloses a process to prepare Tirzepatide employing native chemical ligation (NCL) technique. The NCL method involves a chemo selective reaction of two unprotected peptide segments to produce a transient thioester-linked intermediate and rearranging the obtained thioester-linked intermediate to provide Tirzepatide. Another international patent application WO2021/158444 describes a process for the preparation of Tirzepatide using a solid-phase peptide synthesizer comprising at least two resin reactors in series.

Few more patent applications describe various approaches to prepare Tirzepatide e.g. WO 2023/089594 describes a process involving the coupling of fragments linked to hydrophilic linkers; WO2023/028466 describes a hybrid SPPS/LPPS approach for the preparation of Tirzepatide; CN112592387 A, CN115368452 A describe preparation processes of Tirzepatide using solid phase peptide synthesis; and WO2024134676 describes SPPS approach for the preparation of Tirzepatide involving SPPS synthesis of linear peptide sequence starting with Ser39 till Lys20 followed by sequential growth of Lys-side chain and then continuing the sequential addition of amino acids on the linear backbone, followed by deprotection to get Tirzepatide.

Further, Org. Process Res. Dev. 2021, 25, 1628-1636 discloses a hybrid SPPS/LPPS approach for the preparation of Tirzepatide, which involves coupling of four fragments AA1-14, AA15-21, AA22-29 and AA30-39 in multiple stages to obtain Tirzepatide.

However, the previous syntheses of Tirzepatide suffer from one or the other problems such as an overall low yield, and/or high levels of impurities.

Therefore, there is a need to develop an improved process for preparation of Tirzepatide and purification thereof, which is simple, robust, cost-effective, and avoids or reduces content of impurities.

OBJECTIVE OF THE INVENTION
The objective of the present invention is to provide a process for the synthesis of Tirzepatide.

SUMMARY OF THE INVENTION
In an aspect, the present invention provides a process for the synthesis of Tirzepatide of formula I, which comprises the following steps:
a) preparing a peptide fragment-B,

b) preparing a peptide fragment-C,

c) preparing peptide fragment-A

d) coupling the peptide fragment-B with peptide fragment-C in presence of suitable coupling agents, additives, base and solvents, followed by deprotection of alpha-amino protecting group to provide peptide fragment-D;

e) coupling the peptide fragment-D with the peptide fragment-A in presence of suitable coupling agents, additives, base and solvents to provide peptide of formula- II,

f) deprotecting the peptide of formula II by using suitable deprotecting agents to obtain crude Tirzepatide of formula I.

wherein,
X1 is hydroxy protecting group selected from the group comprising of benzyl (Bzl), tert-butyl (tBu), acetamidomethyl (Acm), and trityl (Trt), tetrahydropyranyl (THP), and 2,5-dichlorobenzyl (Dcb), bromobenzyl (BrBn), 3-pentyl (Pen), tert-butyloxycarbonyl (Boc), cyclohexyl (cHx), tert-butyldiphenylsilyl (TBDPS) and tert-butyldimethylsilyl (TBDMS) group,
X2 is carboxyl protecting group selected from the group comprising of benzyl (Bz), 2,6-dichlorobenzyl (2,6-Cl2Bzl), tert-butyl (tBu), 2,2,2-trichloroethyl (TCE), ß-menthyl (Men) and cyclohexyl (cHx) group,
X3 is an amino protecting group selected from the group comprising of 2-chlorobenzyloxycarbonyl (Cl-Z), tert-butyloxycarbonyl (Boc), 4-methyltrityl (Mtt), Xanthyl (Xan), Trityl (Trt), tert-butyl, benzyl, cyclohexyloxycarbonyl (Hoc), p-methoxybenzyloxycarbonyl (Moz or MeOZ) , 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-ethyl (Dde), 2-(methylsulfonyl)ethoxycarbonyl (Msc), tetrachlorophthaloyl (TCP), p-nitrobenzyloxycarbonyl (pNZ), phenyldisulphanylethyloxycarbonyl (Phdec), phenyldisulphanylethyl oxycarbonyl (Phdec), o-nitrobenzenesulfonyl (o-NBS), allyloxycarbonyl (Alloc) and fluorenylmethyloxycarbonyl (Fmoc) group; and
wherein,
protected side chain moiety at Lys20 ¬is attached to epsilon amino group of Lys and is represented by formula III,

wherein R1 and R2 are same or different carboxyl protecting groups selected from the group comprising of but not limited to primary, secondary and tertiary alkyl, trityl, methoxymethyl, benzyl and allyl.

In another aspect, the present invention provides a process for the preparation of Tirzepatide, which comprises:
a) preparing peptide fragments, A, B, C by solid phase or solution phase syntheses,
b) coupling the peptide fragment-B with peptide fragment-C by using of suitable coupling agents followed by deprotection of alpha-amino protecting group to provide peptide fragment- D,
c) coupling the peptide fragment-D with peptide fragment-A by using suitable coupling agents, to provide peptide of formula-II,
d) deprotecting the peptide of formula II by using suitable deprotecting agents in one or more steps to obtain crude Tirzepatide, and
e) purifying crude Tirzepatide onto RP-HPLC column using silica-based resin and using suitable buffer system to obtain pure Tirzepatide.

In another aspect, the present invention provides peptide compounds of formulae Fragment-A,

Fragment-B,

Fragment-C1,

wherein R3 is H, Fmoc or Boc group

Fragment-D1
wherein R3 is H, Fmoc or Boc group.

BRIEF DESCRIPTION OF ABBREVIATIONS AND DEFININTIONS
The following abbreviations are used throughout the specification and their meaning will be as defined in the table below. Further, the amino acids mentioned in the structural formulae of the present disclosure are represented by their three letter code and unless specifically mentioned, these three letter codes represent L-amino acids.

Aib Aminoisobutyric acid
Boc t-Butyloxycarbonyl
HOBT Hydroxybenzotriazole
HBTU O-(benzotriazol-l-0)-l,l,3,3-tetramethyluronium hexafluorophosphate
ACN Acetonitrile
CTC Chlorotrityl chloride
DPPA Diphenylphosphoryl azide
DCM Dichloromethane
DIC Diisopropylcarbodiimide
DIPEA Diisopropylamine
DMF N, N-dimethylformamide
Fmoc Fluorenylmethyloxycarbonyl
HOBT Hydroxy Benzotriazole
OtBu O-t-Butyl
MTBE Methyl tert-Butyl Ether
t-Bu tert-Butyl
Trt Trityl
TFF Tangential flow filtration
TSTU 2-succinimido-1,1,3,3-tetramethyluronium tetrafluoro borate
HOAt 7-aza-1-hydroxybenzotriazole
TFA Trifluoroacetic acid
TIPS Triisopropylsilane
RP-HPLC Reversed Phase High Performance Liquid Chromatography
LPPS Liquid phase peptide synthesis
SPPS Solid phase peptide synthesis

DETAILED DESCRIPTION OF THE IVENTION
The present invention relates to a process for the preparation of Tirzepatide by coupling of two or more protected peptide fragments.

In one aspect, the present invention provides one more processes to prepare Tirzepatide using novel peptide fragments of formulae: Fragment-A, Fragment-B, Fragment-C1, and Fragment-D1.

Fragment-B,

Fragment-C1,

wherein R3 is H, Fmoc or Boc group

Fragment-D1
wherein R3 is H, Fmoc or Boc group.

In another aspect, the present invention relates to a process for the preparation of Tirzepatide comprising coupling the peptide Fragment-D with the peptide Fragment-A to obtain a compound of formula-II, followed by deprotecting and to obtain Tirzepatide.

In one more aspect, the present invention provides a process for the preparation of Tirzepatide of formula I:

which comprises the steps:
a) preparing a peptide fragment-B,

b) preparing a peptide fragment-C2 by solid phase or solution phase syntheses,

c) deprotecting alpha-amino protection of the peptide fragment-C2 by treating with suitable deprotecting agent to obtain fragment-C

d) coupling of peptide fragment-B with peptide fragment-C in presence of suitable coupling agent, additives, base & solvent to obtained peptide fragment-D2

e) subjecting the peptide fragmen-D2 to deprotection of alpha-amino protecting group using suitable deprotecting reagents to provide peptide fragment-D;

f) preparing peptide fragment-A by solid phase or solution phase synthesis,

g) coupling of the peptide fragment-D with the peptide fragment-A in presence of suitable coupling agent, additives, base & solvent to provide peptide of formula- II,

h) deprotecting the peptide of formula II by using suitable deprotecting agents in one or more steps to obtain Tirzepatide of formula I.
i) purifying crude Tirzepatide onto RP-HPLC column using silica-based resin and using suitable buffer system to obtain pure Tirzepatide.
wherein,
X1 is hydroxy protecting group selected from the group comprising of benzyl (Bzl), tert-butyl (tBu), acetamidomethyl (Acm), and trityl (Trt), tetrahydropyranyl (THP), Cbz, and 2,5-dichlorobenzyl (Dcb), bromobenzyl (BrBn), 3-pentyl (Pen), tert-butyloxycarbonyl (Boc), cyclohexyl (cHx), tert-butyldiphenylsilyl (TBDPS) and tert-butyldimethylsilyl (TBDMS) group, and X1 on different amino acids can be same or different hydroxy protecting group
X2 is carboxy protecting group selected from the group comprising of benzyl (Bz), 2,6-dichlorobenzyl (2,6-Cl2Bzl), tert-butyl (tBu), 2,2,2-trichloroethyl (TCE), ß-menthyl (Men) and cyclohexyl (cHx) group, and X2 on different amino acids can be same or different carboxy protecting group
X3 is an amino protecting group selected from the group comprising of 2-chlorobenzyloxycarbonyl (Cl-Z), tert-butyloxycarbonyl (Boc), 4-methyltrityl (Mtt), Xanthyl (Xan), Trityl (Trt), tert-butyl, benzyl, cyclohexyloxycarbonyl (Hoc), p-methoxybenzyloxycarbonyl (Moz or MeOZ) , 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-ethyl (Dde), 2-(methylsulfonyl)ethoxycarbonyl (Msc), tetrachlorophthaloyl (TCP), p-nitrobenzyloxycarbonyl (pNZ), phenyldisulphanylethyloxycarbonyl (Phdec), phenyldisulphanylethyloxycarbonyl (Phdec), o-nitrobenzenesulfonyl (o-NBS), Allyloxycarbonyl (Alloc) and Fluorenylmethyloxycarbonyl (Fmoc) group; and X3 on different amino acids can be same or different amino protecting group
and wherein,
protected side chain moiety attached to epsilon amino group of Lys20 is represented by formula III:

wherein R1 and R2 are same or different carboxy protecting groups selected from the group comprising of but not limited to primary, secondary, and tertiary alkyl, trityl, methoxymethyl, benzyl and allyl.

In one aspect of the present invention, fragment B is prepared by acylating Lys20 of peptide fragment E-bound to a resin such as CTC-resin, followed by cleaving from the resin to obtain fragment B,

with a protected side chain compound of formula IV,

wherein R1 and R2 are same or different carboxyl protecting groups selected from the group comprising of but not limited to primary, secondary, and tertiary alkyl, trityl, methoxymethyl, benzyl and allyl.

In one more embodiment, fragment B,

is prepared by acylating the resin-bound fragment-F with the protected side chain compound of formula IV or by sequential coupling with AEEAOH (twice), Glu and fatty acid to obtain,

wherein R4 is alpha amino protecting group and X3 is epsilon amino protecting group with proviso that R4 and X3 are different
to obtain acylated fragment G,

and then deprotecting the alpha amino group of Fragment-G and continuing the sequential coupling on the linear backbone followed by cleavage from the resin to obtain the peptide fragment B.

In one more embodiment, fragment B is prepared by sequential SPPS method using Fmoc alpha amino protected amino acids with side chains protected with suitable protecting groups, except at Ly20 position, Fmoc-Lys acylated with the protected fatty acid side chain is used.

In one more aspect, the peptide fragments A, B, C, E, F and G are prepared using solid phase synthesis (SPPS) or solution phase synthesis. In the preferred embodiments, said fragments are prepared by using SPPS method as known in the art using a suitable resin and the suitable coupling agents.

The solid phase synthesis comprises elongation of peptide sequence by coupling protected amino acids onto a peptide resin, cleaving alpha amino protecting group of first amino acid, coupling the second protected amino acid to the first amino acid via a peptide linkage and repeating the cycle until the peptide fragment of desired sequence is obtained.

The resin is selected from the followings: chlorotrityl resin (CTC), 4-methytrityl chloride resin, Rink acid resin, Rink amide resin, PAL resin, Sieber Amide Resin, NovaSyn TGT resin, HMPB-AM resin.

The resin used for the synthesis of peptide fragments A and B are same or different resins selected from the group comprising of chlorotrityl resin (CTC), 4-methytrityl chloride resin, Rink acid resin. The precursors of peptide fragment C are prepared by SPPS method using resins such as: chlorotrityl resin (CTC), 4-methytrityl chloride resin, and rink acid resin are subjected to the amidation by conventional methods to obtain peptide fragment C. Alternatively, peptide fragment C is prepared by SPPS method using amide resins selected from Rink amide resin, PAL resin, Sieber Amide Resin, and preferably Sieber amide resin.

In one more embodiment of the present invention, the process to prepare Tirzepatide involves coupling of three peptide fragments in two stages to obtain protected Tirzepatide followed by deprotection of the protected Tirzepatide to obtain Tirzepatide. The process for the preparation of linear 1-39 peptide involves coupling of peptide fragment B (16 amino acids) with peptide fragment C (11 amino acids) in a solution in the presence of one or more coupling agents and one or more base followed by deprotection of alpha-amino protecting group to provide peptide fragment-D (27 amino acids). In the second stage, the obtained peptide fragment D (27 amino acids) is coupled with a peptide fragment A (12 amino acids) in a solution in the presence of one or more coupling agent and one or more base to give a protected Tirzepatide, which is then deprotected with suitable deprotecting agents to produce Tirzepatide.

The coupling reagent is used in the presence of or absence of additives. The coupling reagents includes but are not limited to N,N'-Diisopropylcarbodiimide (DIC), dicyclohexyl carbodiimide (DCC), Hexafluorophosphate Azabenzotriazole Tetramethyl Uronium (HATU), O-(benzotriazol-l-0)-l,l,3,3-tetramethyluronium hexafluorophosphate (HBTU), 2-(1H-Benzotriazole-1-y (TBTU) , benzotriazolyl-N-oxytrisdimethylaminophosphonium hexafluorophosphate (BOP), Bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BOP-Cl), benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), Bromotri(pyrrolidino)phosphonium hexafluorophosphate (PyBrOP), Isobutyl carbonochloridate (IBCF), 1-(dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (WSCDl), 2-Ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ), Isopropyl chloroformate (IPCF), 2-(5-Norbornene-2,3-dicarboximido)-1,1,3,3-tetramethyluronium tetrafluoroborate (TNTU), Propanephosphonic acid anhydride (PPAA), 2-Succinimido-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU), chlorotri(pyrrolidino)phosphonium hexafluorophosphate (PyClOP) and mixture thereof.

The additive includes but are not limited to N-Hydroxybenzotriazole (HOBt), 4-Dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HODhbt), 7-aza-1-hydroxybenzotriazole (HOAt), 1-Hydroxy-6-(trifluoromethyl) benzotriazole (6-CF3- HOBt), ethyl-2-cyano-2-(hydroxyimino) acetate (Oxyma) and mixture thereof.

The base used for coupling is selected from the organic or inorganic bases. The inorganic base is selected from the group comprising of potassium carbonate, lithium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, sodium hydroxide, potassium hydroxide, ammonium hydroxide, and mixtures thereof; the organic base comprises t-butylamine, 4-Dimethylaminopyridine (DMAP), diisopropylamine, ?,?-diisopropylethylamine triethylamine, dimethylamine, trimethyl amine, isopropyl ethylamine, pyridine, N-methyl morpholine, piperidine, l,8-diazabicyclo[5.4.0]undec-7-ene (DBU), and/or mixtures thereof. The solvent used for coupling reaction is selected from the group comprising of but not limited to dimethylformamide (DMF), dimethylsulfoxide (DMSO), N-Methyl pyrrolidine (NMP), Dimethylacetamide (DMAC), dichloromethane (DCM), methanol, isopropanol, dichloroethane, 1,4-dioxane, tetrahydrofuran (THF), 2-methyl tetrahydrofuran, ethyl acetate, acetonitrile, acetone and mixtures thereof.

The base used for the deprotection of Fmoc-protected alpha amino group includes but is not limited to t-butylamine, piperidine, diethyl amine, DBU, piperazine, pyrrolidine, derivatives thereof in presence of solvent selected from the group comprising of but not limited to alcohol, amide and ether and mixture thereof.

In one more embodiment, if there is an unreacted N-terminal amine following a coupling reaction, then second coupling is performed. In one more embodiment an unreacted amine after the coupling is capped with capping agents like acetic anhydride.

In one more embodiment, the cleavage of resin bound precursors of fragments A, B, C, and E from the resin is performed by treating the resin linked fragments with of trifluoroactetic acid (TFA), 1,2-ethanedithiol (EDT), dimethyl sulfide (DMS), thioanisole, phenol, anisole and mixture thereof in a suitable solvent. The solvent is selected from the solvents such as alcohol, amide, ether DCM and DMF.

In one more embodiment, peptide of formula II is subjected to Fmoc deprotection by treating with a base in suitable solvents to get Fmo-deprotected successor, which is further subjected to the global deprotection of all amino acids side chains. The base used for the Fmoc deprotection is selected from the group comprising of t-butylamine, piperidine, diethyl amine, DBU, piperazine, pyrrolidine, derivatives thereof and mixtures thereof. The solvent used in the Fmoc deprotection is selected from the group comprising of but not limited to alcohol, amide and ether.

In one embodiment, Fmoc deprotected successor of peptide of formula II is subjected to the side chain deprotection i.e. global deprotection using deprotecting agents selected from the group comprising of trifluoroactetic acid (TFA), 1,2-ethanedithiol (EDT), dimethyl sulfide (DMS), thioanisole, phenol, anisole and mixture thereof. The deblocking is preferably carried out in a cocktail mixtures such as TFA: TIPS:DTT:solvent (or) TFA: TIPS:DTT:water:solvent (or) TFA:solvent. The solvent is selected from the group comprising of water, dimethyl sulfoxide, alcohols selected form methanol, ethanol, 1-propanaol, isopropanol, n-butanol; chlorinated solvents selected form dichloromethane, dichloroethane, chlorobenzene; ether solvents selected form diethyl ester, tetrahydrofuran, diisopropyl ether and or combination thereof. The additional cocktail reagents are selected from 1,2-ethanedithiol (EDT), dimethyl sulfide (DMS), thioanisole, phenol, anisole etc. In an embodiment, the present invention provides deblocking of protected linear peptide using a mixture of TFA: DCM or piperidine: DMF.

After completion of cleavage, the linear peptide or its suitable salt such as TFA or acetate salt is precipitated by using suitable solvent, which is selected from ether solvent like diethyl ether, dioxane, diisopropyl ether, methyl tertiary butyl ether, and tetrahydrofuran or ethyl acetate to afford desired peptides fragments.

In one more embodiment, the crude Tirzepatide is enriched by tangential flow filtration and / or purified using preparative column chromatography or reverse phase column chromatography (RP-HPLC) using suitable buffer system.

In yet another aspect of the present invention provides a process for the purification of Tirzepatide of formula I, which comprises:
a) purification of crude Tirzepatide onto reverse phase column chromatography (RP-HPLC) with suitable buffer system; and
b) lyophilization to obtain pure Tirzepatide.

The column used for purification of Tirzepatide may be conventional column known in the art. The column in preparative HPLC may be packed with reverse phase C18 hybrid silica. Suitable silica gel types, which can be selected from, but are not limited to the following silica gel sorbents: Kromasil™C18 100 - 16, Kromasil™C18 100 - 10, Kromasil™C8 100 - 16, Kromasil™C4 100 - 16, Kromasil™ Phenyl 100 - 10, Kromasil™ CI 8 Eternity 100 - 5, Kromasil™ C4 Eternity 100 - 5, Chromatorex™ CI 8 SMB 100-15 HE, Chromatorex™ C8 SMB 100-15 HE, Chromatorex™ C4 SMB 100-15 HE, Daisopak™ SP 120-15 ODS-AP, Daisopak™ SP 120-10-C4-Bio, Daisopak™ SP 200-10-C4-Bio, Zeosphere™ C18 100-15, Zeosphere™ C8 100-15, Zeosphere™ C4 100-15, SepTech ST 150-10 C18, Luna C18 100-10, Diasogel C8 80 mm column, Gemini C18 110-10, YMC Triart C18 120-5 and YMC Triart C8 200-10. Buffer system selected from but not limited to ammonium bicarbonate buffer (pH 9.0 – 9.5, pH), Triethylammonium phosphate, Ammonium Chloride buffer (pH 7.5), Tris HCl, TEAP, acetonitrile, TFA & Water.

Tirzepatide as produced by any one of the reaction conditions and purification process described above is isolated in an amorphous or crystalline state. Purified Tirzepatide further optionally undergoes lyophilization to provide amorphous Tirzepatide.

Tirzepatide prepared using above-described process of the present invention results in high yield, particularly consistent with high purity. In the process according to the present invention, one or more of the different factors relating to the reagents and their quantities which are used in carrying out the various steps to prepare Tirzepatide; the manner in which the steps are carried out; the methods used; and the various process parameters like temperature, pH, concentration, etc. were optimized and controlled in proper manner so as to obtain the desired product in a consistent manner.

Having described the invention with reference to certain aspects and embodiments, which will become apparent to one skilled in the art from consideration of the specification. The invention is further defined by reference to the following examples. It will be apparent to those skilled in the art that many modifications, both to materials and methods, may be practiced without departing from the scope of the invention.

EXAMPLES

EXAMPLE 1: Synthesis of Fmoc-Tyr(tBu)1-Aib2-Glu(tBu)3-Gly4-Thr(tBu)5-Phe6-Thr(tBu)7-Ser(tBu)8-Asp(tBu)9-Tyr(tBu)10-Ser(tBu)11-Ile12-COOH (Peptide Fragment A)
Chlorotrityl resin (35.0 g) was taken into SPPS glass reaction vessel and swollen with DCM (3-10 Volumes) for 20-30 min. Fmoc-Ile12-OH (1.0-2.5 eq.), and DIPEA (2.0-6.0 eq.) were dissolved in DCM (3-10 Volumes), and kept under cooled condition in ice-water bath. The amino acid was then added to CTC resin and stirred for 2-4 h at room temperature, followed by wash with DCM (3-10 Volumes) and dried. The loading of resulting resin was measured by UV analytical method. The unreacted resin after first coupling was capped with DIPEA (1%) in 1:1 v/v solution of Methanol: DCM (3-10 Volumes) and then washed with DCM (3-10 Volumes). Fmoc deprotection was undertaken using a 20% piperidine in DMF, for 5-30 minutes each. Fmoc protected amino acid couplings were carried out sequentially using 2-3 equivalents of each amino acid, 2-4 equivalents of HOBt/DIC. The resin was washed with thrice with DMF (3-10 Volumes), followed by twice with IPA (3-10 Volumes), followed by twice with DCM (3-10 Volumes) then thrice with DMF (3-10 Volumes) after each coupling. The resin was dried under vacuum for 12-16 h.
Deprotection and Cleavage:
20 mL of the cleavage cocktail made with 1% trifluoroacetic acid (TFA) in DCM was added to the dried peptidyl resin (Fmoc-Tyr(tBu)1-Aib2-Glu(tBu)3-Gly4-Thr(tBu)5-Phe6-Thr(tBu)7-Ser(tBu)8-Asp(tBu)9-Tyr(tBu)10-Ser(tBu)11-Ile12- Resin) and mixed for 15 min at 5° C and solution drained into a RBF. The solution obtained from cleavage reaction was washed with water (0.5-3 volumes w.r.t organic), followed by brine (0.5-3 volumes w.r.t organic) and the organic layer was dried on anhydrous sodium sulfate followed by rotary evaporation to obtain crude solid. The crude solid slurry was then washed with MTBE to obtain precipitated of protected Tirzepatide fragment A (1-12) was then filtered and dried under vacuum.
[M+H] + Calculated mass: 1589.66; Observed mass: 1589.63. (after global deprotection)
Yield 65%;
Purity: 88.9%.

EXAMPLE 2: Synthesis of Fmoc-Aib13-Leu14-Asp(tBu)15-Lys (Boc)16-Ile17-Ala18-Gln(Trt)19-Lys(Protected side chain)20-Ala21-Phe22-Val23-Gln(Trt)24-Trp(Boc)25-Leu26-Ile27-Ala28-COOH (Fragment B)

CTC resin (5.2 g) was taken into SPPS glass reaction vessel and swollen with DCM (3-10 Volumes) for 20-30 min. Fmoc-Ala28-OH (1.0-2.0 eq.), and DIPEA (2.0-4.0 eq.) were dissolved in DCM (3-10 Volumes), and kept under cooled condition in ice-water bath. The activated amino acid was added to CTC resin and stirred for 2-4 h at room temperature. After 2-4 hours the resin was washed with DCM (3-10 Volumes) and dried. The loading of resulting resin was measured by UV analytical method. The unreacted resin after first coupling was capped with DIPEA (1%) in 1:1 v/v solution of Methanol: DCM (3-10 Volumes), stirred for 30 min. after 30 min resin washed with DCM (3-10 Volumes). Fmoc deprotection was undertaken using a 20% piperidine in DMF, for 5-30 minutes each. Fmoc protected amino acid couplings were carried out sequentially out using 2-3 equivalents of amino acid, 2-4 equivalents of HOBt DIC involve use of Fmoc protected lysine attached to side chain [Lys(side chain)-OH] coupling. The resin was washed with thrice DMF (3-10 Volumes), followed by twice with IPA (3-10 Volumes), followed by twice with DCM (3-10 Volumes) then thrice with DMF (3-10 Volumes) after each coupling. The resin was dried under vacuum for 12-16hr.
Deprotection and Cleavage:
20 mL of the cleavage cocktail made with 1% trifluoroacetic acid (TFA) in DCM was added to the dried peptidyl resin (Fmoc-Aib13-Leu14-Asp(tBu)15-Lys(Boc)16-Ile17-Ala18-Gln(Trt)19-Lys(Protected side chain)20-Ala21-Phe22-Val23-Gln(Trt)24-Trp(Boc)25-Leu26-Ile27-Ala28-CTCResin) and mixed for 15 min at 5° C and solution drained into a RBF. The solution obtained from cleavage reaction was washed with water (0.5-3 volumes w.r.t organic), followed by brine (0.5-3 volumes w.r.t organic) and the organic layer was dried on anhydrous sodium sulfate followed by rotary evaporation to give crude solid. The crude solid was slurry washed with MTBE to obtain precipitated of protected Tirzepatide fragment B (13-28) was then filtered and dried under vacuum.
[M+H] + Calculated mass: 2794.59; Observed mass: 2794.55 (after global deprotection).
Yield: 60%
Purity: 66.87%

EXAMPLE 3: Synthesis of Fmoc-Gly29-Gly30-Pro31-Ser(tBu)32-Ser(tBu)33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser(tBu)39-NH2 (Fragment C2)

Sieber Amide resin (25 g) was taken into SPPS glass reaction vessel and swollen with DCM (3-10 Volumes) for 20-30 min. Fmoc-Ser(tBu)39-OH (1.0-4.0 eq.), and HOBt (2.0-4.0 eq.) & DIC (2.0-4.0 eq.) were dissolved in DCM (3-10 Volumes), at RT. The activated amino acid was added to CTC resin and stirred for 2-4 h at room temperature. After 2-4 hours the resin was washed with DCM (3-10 Volumes) and dried. The loading of resulting resin was measured by UV analytical method.

The unreacted resin after first coupling was capped with Acetic anhydride (3 eq.), DIPEA (1%) in DCM (3-10 Volumes), stirred for 30 min. after 30 min resin washed with DCM (3-10 Volumes). Deprotection was undertaken using a 20% piperidine in DMF, for 5-30 minutes each. Fmoc protected amino acid couplings were carried out sequentially using 2-3 equivalents of amino acid, 2-4 equivalents of HOBt DIC, in DMF under cold condition. The resin was washed with thrice DMF (3-10 Volumes), followed by twice with IPA (3-10 Volumes), followed by twice with DCM (3-10 Volumes) then thrice with DMF (3-10 Volumes) after each coupling. The resin was dried under vacuum for 12-16hr.
Deprotection and Cleavage:
20 mL of the cleavage cocktail made with 1% trifluoroacetic acid (TFA) in DCM was added to the dried peptidyl resin peptidyl resin (Fmoc-Gly29-Gly30-Pro31-Ser(tBu)32-Ser(tBu)33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser(tBu)39-Resin) and mixed for 15 min at 5° C and solution drained into an RBF. The solution obtained from cleavage reaction was washed with water (0.5-3 volumes w.r.t organic), followed by brine (0.5-3 volumes w.r.t organic) and the organic layer was dried on anhydrous sodium sulfate followed by rotary evaporation to give crude solid. The crude solid was slurry washed with MTBE to obtain precipitated of protected Tirzepatide fragment C2 (29-39) Fmoc protected was then filtered and dried under vacuum.
[M+H] + Calculated mass: 1131.50; Observed mass: 1131.52.(mass is after global cleavage)
Yield: 70%.
Purity: 89.89 %.

EXAMPLE 4: Synthesis of Gly29-Gly30-Pro31-Ser(tBu)32-Ser(tBu)33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser(tBu)39-NH2 (Fragment C)
The obtained peptide fragment C2 (29-39) (1.0 eq.) Fmoc protected subjected to Fmoc deprotection using 20% piperidine in DMF in presence of base Ter-Butyl amine (10-50 eq.) / DCM at 0 °C. The reaction was kept at RT for 8-24 hours after the solvent evaporation and drying under vacuum to give crude solid to give of fragment C (29-39).
Yield: 80 %.
Purity: 91.93%

EXAMPLE 5: Synthesis of fragment D2: Fmoc-Aib13-Leu14-Asp(X2)15-Lys(X3)16-Ile17-Ala18-Gln(X3)19-Lys (Protected side chain)20-Ala21-Phe22-Val23-Gln(X3)24-Trp(X3)25-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser(X1)32-Ser(X1)33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser(X1)39NH2
The obtained peptide fragment C (29-39) (0.5-2.0 eq.) coupled to peptide fragment B (13-28) (0.5-2.0 eq.) DCM solution in presence of HOAt (0.5-3.0 eq.) in DMF & HBTU (1.0-5.0 eq.) and followed by DIPEA (1.0-5.0 eq.) at cold condition then reaction mixture kept RT for 1-24 hr. after completion of reaction quenched with water to precipitate Fragment D (13-39) as white solid. The precipitate was filtered and washed thrice with water (3-10 volumes) and the solid was dried under vacuum for 4-24 h. To obtained peptide fragment D2 Fmoc-Aib13-Leu14-Asp(X2)15-Lys(X3)16-Ile17-Ala18-Gln(X3)19-Lys (Protected side chain)20-Ala21-Phe22-Val23-Gln(X3)24-Trp(X3)25-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser(X1)32-Ser(X1)33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser(X1)39-NH2
[M+H] + Calculated mass: 3683.91; Observed mass: (after global deprotection)
Yield: 60% ;
Purity: 80%.

EXAMPLE 6: Synthesis of fragment D: Aib13-Leu14-Asp(X2)15-Lys(X3)16-Ile17-Ala18-Gln(X3)19-Lys (Protected side chain)20-Ala21-Phe22-Val23-Gln(X3)24-Trp(X3)25-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser(X1)32-Ser(X1)33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser(X1)39-NH2
The obtained peptide fragment D2 (1.0 eq.) was subjected to Fmoc deprotection using 20 % piperidine in DMF in presence of tert-Butyl amine (10-50 eq.) in DCM as base to give the peptide fragment D (13-39).
Yield: 70 %; Purity: 60%.

EXAMPLE 7: Synthesis of Tirzepatide (Formula-I)
Step 1: Preparation of protected Tirzepatide (Formula-II):
The obtained peptide fragment D (13-39) (0.5-2.0 eq.) was added to peptide fragment A (1-12) (0.5-2.0 eq.) DMF solution in presence of HOAt (0.5-3.0 eq.in DMF & HBTU (1.0-5.0 eq.) followed by DIPEA (1.0-5.0 eq.) at cold condition then reaction mixture kept RT for 1-24 hr. after completion of reaction quenched with water to precipitate protected Tirzepatide of formula II as white solid. The precipitate was filtered and washed thrice with water (3-10 volumes) and the solid was dried under vacuum for 4-24 h.
Step 2: Global cleavage:
The obtained peptide formula II from step 1 were treated with TBA (20 eq.) in DCM (3-10 volumes) for 1-24 hours, followed by precipitation using MTBE to obtained Fmoc deprotected compound of formula II. The Fmoc deprotected compound of formula II were dissolved in cleavage cocktail (TFA:TIPS:EDT:Phenol:H2O::90:2.5:2.5:2.5:2.5) in cooled condition, stirred at room temperature for 3 h. After 3h the solution was filtered and precipitated by the addition of 10X volumes of MTBE. Further the precipitate was washed twice with 10X volumes of acetone followed by 10X volumes of MTBE for two times. The obtained precipitated product is a TFA salt of Tirzepatide.
M+H]+ Calculated mass:4811.52 Observed mass: 4811.47
Yield: 20%; Purity: 55%.

EXAMPLE 8: Purification and Lyophilization:
Stage-1: The crude Tirzepatide (TFA salt of Tirzepatide) was dissolved in 20 mM Ammonium bicarbonate buffer, (pH 9.0 – 9.5), and purified using RP-HPLC with wavelength 220 nm, Diasogel C8 80 mm column of C8 reverse phase column, mobile phase A: Ammonium Chloride buffer (20 mM and pH 7.5) and mobile phase B: 70% acetonitrile and 30% buffer A, with flow rate: 128 mL/min, purity >85% then loaded to column for second purification.

Stage-2: The obtained fractions from stage 1 were passed through Waters RP-HPLC system, wavelength 220 nm, Diasogel C8 80 mm column of C8 reverse phase column, with mobile phase A: 0.01% TFA in water and mobile phase B: acetonitrile with flow rate: 128 mL/min. > 99.0% were collected, pooled together, concentrated using rotary evaporation and lyophilized to give pure Tirzepatide.
,CLAIMS:We Claim

1. A method of preparing Tirzepatide of formula-I or a pharmaceutically acceptable salt thereof, the method comprising:

a) preparing peptide fragment-A: Fmoc-Tyr(X1)1-Aib2-Glu(X2)3-Gly4-Thr(X1)5-Phe6-Thr(X1)7-Ser(X1)8-Asp(X2)9-Tyr(X1)10-Ser(X1)11-Ile12-COOH,
b) preparing fragment-B: Fmoc-Aib13-Leu14-Asp(tBu)15-Lys (Boc)16-Ile17-Ala18-Gln(Trt)19-Lys (Protected side chain)20-Ala21-Phe22-Val23-Gln(Trt)24-Trp(Boc)25-Leu26-Ile27-Ala28-COOH,
c) preparing fragment-C: Gly29-Gly30-Pro31-Ser(tBu)32-Ser(tBu)33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser(tBu)39-NH2
d) coupling the peptide fragment-B with the peptide fragment-C in presence of coupling agent, additive, base and solvent to obtain peptide fragment-D2: Fmoc-Aib13-Leu14-Asp(X2)15-Lys(X3)16-Ile17-Ala18-Gln(X3)19-Lys (Protected side chain)20-Ala21-Phe22-Val23-Gln(X3)24-Trp(X3)25-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser(X1)32-Ser(X1)33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser(X1)39-NH2;
e) deprotecting the alpha-amino protection of fragment-D2 using deprotecting agent to obtain peptide fragment-D: Aib13-Leu14-Asp(X2)15-Lys(X3)16-Ile17-Ala18-Gln(X3)19-Lys (Protected side chain)20-Ala21-Phe22-Val23-Gln(X3)24-Trp(X3)25-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser(X1)32-Ser(X1)33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser(X1)39-NH2;
f) coupling the peptide fragment-D with peptide fragment-A in presence of coupling agent, additive, base and solvent to obtain protected 1-39 fragment compound of formula II:
Fmoc-Tyr(X1)1-Aib2-Glu(X2)3-Gly4-Thr(X1)5-Phe6-Thr(X1)7-Ser(X1)8-Asp(X2)9-Tyr(X1)10-Ser(X1)11-Ile12-Aib13-Leu14-Asp(X2)15-Lys(X3)16-Ile17-Ala18-Gln(X3)19-Lys(Protected side chain)20-Ala21-Phe22-Val23-Gln(X3)24-Trp(X3)25-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser(X1)32-Ser(X1)33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser(X1)39-NH2
and
g) deprotecting the alpha-amino protection and side chain protections of the peptide of formula-II, using deprotecting agent to obtain Tirzepatide of formula I
wherein, X1 represents hydroxy protecting group; X2 represents carboxyl protecting group and X3 represents an amino protecting group.

2. The process according to claim 1, wherein the coupling agent is selected from the group comprising of DIC, DCC, HATU, HBTU, TBTU, BOP, BOP-Cl, PyBOP, PyBrOP, IBCF, WSCDl, EEDQ, IPCF, TNTU, PPAA, TSTU, PyClOP, Oxyma pure, TCTU, COMU, HOSu and mixtures thereof.

3. The process according to claim 1, wherein the additive is selected from the group comprising of HOBt, HODhbt, HOAt, 6-CF3-HOBt, 6-?O2-?OBt, ethyl-2-cyano-2-(hydroxyimino) acetate (Oxyma) and mixtures thereof.

4. The process according to claim 1, wherein the base is selected from the group comprising of t-butylamine, 4-Dimethylaminopyridine (DMAP), diisopropylamine, ?,?-diisopropylethylamine, triethylamine, dimethylamine, trimethyl amine, isopropyl ethylamine, pyridine, N-methyl morpholine, piperidine, l,8-diazabicyclo[5.4.0]undec-7-ene (DBU), potassium carbonate, lithium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, sodium hydroxide, potassium hydroxide, ammonium hydroxide, and mixtures thereof.

5. The process as claimed in claim 1, wherein the solvent for coupling is selected from the group comprising of dimethylformamide (DMF), dimethyl sulfoxide (DMSO), N-Methyl pyrrolidine (NMP), Dimethylacetamide (DMAC), dichloromethane (DCM), methanol, isopropanol, dichloroethane, 1,4-dioxane, tetrahydrofuran (THF), 2-methyl tetrahydrofuran, ethyl acetate, acetonitrile, acetone and mixtures thereof.

6. The process as claimed in claim 1, wherein the deprotecting agent for alpha-amino deprotection is selected from the group comprising of t-butylamine, piperidine, diethyl amine, DBU, piperazine, pyrrolidine, derivatives thereof and mixtures thereof.

7. The process as claimed in claim 1, wherein the deprotecting agent for deprotection of side chains of amino acids is selected from the group comprising of trifluoroactetic acid (TFA), 1,2-ethanedithiol (EDT), dimethyl sulfide (DMS), thioanisole, phenol, anisole and mixtures thereof.

8. The process as claimed in claim 1, wherein the solvent for deprotection is selected from the group comprising of water, dimethyl sulfoxide, alcohols selected form methanol, ethanol, 1-propanaol, isopropanol, n-butanol; chlorinated solvents selected form dichloromethane, dichloroethane, chlorobenzene; ether solvents selected form diethyl ester, tetrahydrofuran, diisopropyl ether and mixtures thereof.

9. The process according to claim 1, wherein the peptide fragments A, B and C are prepared by solid phase peptide synthesis; and the peptide fragment-D and peptide of formula-II are prepared in solution phase.

10. A compound or a pharmaceutical salt or solvates thereof selected from group comprising of:



and

Wherein,
X1 represents hydroxy protecting group
X2 represents carboxyl protecting group
X3 represents an amino protecting group
R3 is H, Fmoc or Boc.