DESC:RELATED APPLICATION:
This application claims the benefit of the filing date of Indian Provisional Patent Application No. 202321053066 filed on Aug. 08, 2023 and Indian Provisional Patent Application No. 202321053067 filed on Aug. 08, 2023.

FIELD
The present invention relates to an efficient process for the preparation of Tirzepatide, or a pharmaceutically acceptable salt thereof. The present invention also relates to novel fragments as intermediates and use thereof in the preparation of Tirzepatide.

BACKGROUND
Tirzepatide (Figure 1) is a novel glucose-dependent insulinotropic polypeptide (GIP) receptor and glucagon-like peptide-1 (GLP-1) receptor agonist indicated as an adjunct to diet and exercise to improve glycemic control in adults with type 2 diabetes mellitus. Tirzepatide is a 39-amino acid synthetic peptide. It consists of a 39 amino acid peptide backbone along containing 2 non-coded amino acids (aminoisobutyric acid, Aib) in positions 2 and 13, a C-terminal amide, and Lys residue at position 20 is attached to 1,20-eicosanedioic acid via a linker which consists of a ?-Glu and two [2-(2-aminoethoxy)ethoxy]acetic acid. [AEEA]. Tirzepatide is represented by the structure of formula I as below:

Tyr1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Ile12-Aib13-Leu14-Asp15-Lys16-Ile17-Ala18-Gln19-Lys20[AEEA-AEEA-?-Glu-C20-diacid]-Ala21-Phe22-Val23-Gln24-Trp25-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser32-Ser33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39
Formula-I

Tirzepatide was first disclosed in US 9,474,780 (the ‘780 patent”). The ‘780 patent describes a method for the preparation of Tirzepatide by using standard Fmoc-/tBu solid-phase peptide synthesis protocols on an automated peptide synthesizer. An orthogonal protecting group strategy was employed to allow site-specific conjugation of the fatty acid moiety to K20 following synthesis of the linear sequence. Cleavage from the resin, and purification by reverse-phase high performance liquid chromatography afforded Tirzepatide.
WO2020159949, CN110903355, CN112110981, CN112592387, CN115181173, CN115181174, CN115651075, CN115991742, CN115160429, CN116120403, CN116178523, WO2023089594, WO2024112617 discloses different SPPS/fragment based approach to prepare Tirzepatide.
In the synthesis of large peptide molecules, such as Tirzepatide, the conformation of the growing peptide chain and its physico-chemical properties are of considerable importance. The formation of secondary structures often leads to problems of aggregation causing incomplete coupling reactions, resulting in a decrease in the synthetic yield and purity of the final compound.
Therefore, there remains a continuing need for preparation of Tirzepatide in better yields, more purity and has low impurities; and which is commercially feasible as well.
The present invention describes a process for the preparation of Tirzepatide or a pharmaceutically acceptable salt thereof involving use of new fragments as intermediate.
The inventors of the present invention have found that Tirzepatide or a pharmaceutically acceptable salt thereof, as obtained according to the process of present invention possesses desirable properties when formulated as a pharmaceutical composition.

SUMMARY
First aspect of the present invention relates to an improved process of preparation of Tirzepatide comprising;
a. loading of Fmoc protected Ca-carboxamide Serine i.e. Fmoc-Ser39-NH2 to a resin solid-phase support in the presence of coupling agent, additive and base;
b. deprotection of Fmoc protecting group using deprotecting agent and sequential coupling of amino acids with N-terminal protection and side chain orthogonal protection based on the sequence of peptide backbone of Tirzepatide till Tyr1 in the presence of coupling agent, additive and base to obtain SEQ ID: 1, wherein Xn is selected from the group consisting of Alloc, Mtt, Dde, Mmt and ivDde;
c. deprotection of protecting group (Xn) from Lys20 to obtain SEQ ID: 2 and converting the resulting peptide to Tirzepatide.
Second aspect of the present invention relates to an improved process of preparation of Tirzepatide comprising;
a. loading of Fmoc protected serine to a resin solid-phase support in the presence of coupling agent;
b. deprotection of Fmoc protecting group using deprotecting agent and sequential coupling of amino acids with N-terminal protection and side chain protection based on the sequence of peptide backbone of Tirzepatide till Ser8 in the presence of coupling agent; to obtain Fmoc-Ser8-Asp9-Tyr10-Ser11-Ile12-Aib13-Leu14-Asp15-Lys16-Ile17-Ala18-Gln19-Lys20(Xn)-Ala21-Phe22-Val23-Gln24-Trp25-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser32-Ser33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39-resin;
c. deprotection of Fmoc protecting group using deprotecting agent followed by coupling of SEQ ID: 4 with in the presence of coupling agent; to obtain SEQ ID: 6;
d. deprotection of N-terminal protecting group using deprotecting agent followed by coupling of SEQ ID: 5 in the presence of coupling agent; to obtain SEQ ID: 7;
e. deprotection of N-terminal protecting group using deprotecting agent followed by sequential coupling of amino acids with N-terminal protection on the sequence of peptide backbone of Tirzepatide till Tyr1 in the presence of coupling agent; to obtain SEQ ID: 8;
f. deprotection of protecting group (Xn) from Lys20 and reacting with side chain protected or un-protected fragment eicosanedioic acid-?-Glu-AEEA-AEEA-OH OR sequentially reacting the resin with AEEA, AEEA, ?-glutamic acid(OtBu), eicosanedioic acid(OtBu) obtain SEQ ID: 9; and converting the resulting peptide to Tirzepatide.
Third aspect of the present invention relates to an improved process of preparation of Tirzepatide comprising Fmoc-Aib2-Glu3-Gly4-Thr5-OH (SEQ ID: 10)
Fourth aspect of the present invention relates to an improved process of preparation of Tirzepatide comprising Tyr1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Ile12-Aib13-Leu14-Asp15-Lys16-Ile17-Ala18-OH (SEQ ID: 11).
Fifth aspect of the present invention relates to an improved process of preparation of Tirzepatide comprising Lys16-Ile17-Ala18-OH (SEQ ID: 12).
In further aspect, the present invention relates to a compound of SEQ ID: 1, SEQ ID: 2, SEQ ID: 3, SEQ ID: 4, SEQ ID: 5, SEQ ID: 6, SEQ ID: 7, SEQ ID: 8, SEQ ID: 9, SEQ ID: 10, SEQ ID: 11, SEQ ID: 12(as tabulated below) and process of preparation thereof and its use in the preparation of Tirzepatide.
Tyr1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Ile12-Aib13-Leu14-Asp15-Lys16-Ile17-Ala18-Gln19-Lys20(Xn)-Ala21-Phe22-Val23-Gln24-Trp25-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser32-Ser33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39(Resin)-NH2 SEQ ID: 1
Tyr1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Ile12-Aib13-Leu14-Asp15-Lys16-Ile17-Ala18-Gln19-Lys20-Ala21-Phe22-Val23-Gln24-Trp25-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser32-Ser33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39(Resin)-NH2 SEQ ID: 2
Tyr1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Ile12-Aib13-Leu14-Asp15-Lys16-Ile17-Ala18-Gln19-Lys20(AEEA-AEEA-?-Glu(OtBu)-C20-diacid(OtBu))-Ala21-Phe22-Val23-Gln24-Trp25-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser32-Ser33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39(Resin)-NH2 SEQ ID: 3
Phe6-Thr7[Psi(Me, Me)Pro]-OH SEQ ID: 4
Gly4-Thr5[Psi(Me, Me)Pro]-OH SEQ ID: 5
Phe6-Thr7[Psi(Me,Me)Pro]-Ser8-Asp9-Tyr10-Ser11-Ile12-Aib13-Leu14-Asp15-Lys16-Ile17-Ala18-Gln19-Lys20-Ala21-Phe22-Val23-Gln24-Trp25-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser32-Ser33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39 SEQ ID: 6
Gly4-Thr5[Psi(Me,Me)Pro]-Phe6-Thr7[Psi(Me,Me)Pro]-Ser8-Asp9-Tyr10-Ser11-Ile12-Aib13-Leu14-Asp15-Lys16-Ile17-Ala18-Gln19-Lys20-Ala21-Phe22-Val23-Gln24-Trp25-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser32-Ser33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39 SEQ ID: 7
Tyr1-Aib2-Glu3-Gly4-Gly4-Thr5[Psi(Me,Me)Pro]-Phe6-Thr7[Psi(Me,Me)Pro]-Ser8-Asp9-Tyr10-Ser11-Ile12-Aib13-Leu14-Asp15-Lys16-Ile17-Ala18-Gln19-Lys20-Ala21-Phe22-Val23-Gln24-Trp25-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser32-Ser33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39 SEQ ID: 8
Tyr1-Aib2-Glu3-Gly4-Gly4-Thr5[Psi(Me,Me)Pro]-Phe6-Thr7[Psi(Me,Me)Pro]-Ser8-Asp9-Tyr10-Ser11-Ile12-Aib13-Leu14-Asp15-Lys16-Ile17-Ala18-Gln19-Lys20(AEEA-AEEA-?-Glu(OtBu)-C20-diacid(OtBu))-Ala21-Phe22-Val23-Gln24-Trp25-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser32-Ser33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser3 SEQ ID: 9
Aib2-Glu3-Gly4-Thr5 SEQ ID: 10
Tyr1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Ile12-Aib13-Leu14-Asp15-Lys16-Ile17-Ala18 SEQ ID: 11
Lys16-Ile17-Ala18 SEQ ID: 12
wherein the terminal amino acids are free, resin bound or protected with a suitable protecting group; and wherein, the side chain of amino acids are free or protected with a suitable protecting group.
In another aspect, the present invention relates to a compound wherein the compound is any one of the amino acid sequences selected from the group consisting of SEQ ID: 1, SEQ ID: 2, SEQ ID: 3, SEQ ID: 6, SEQ ID: 7, SEQ ID: 8, SEQ ID: 9, SEQ ID: 10, SEQ ID: 11 or a pharmaceutically acceptable salt thereof.
In yet another aspect, the present invention relates to a process for the preparation of Tirzepatide or a pharmaceutically acceptable salt thereof, comprising use of polypeptide or a pharmaceutically acceptable salt thereof selected from compound of SEQ ID: 1, SEQ ID: 2, SEQ ID: 3, SEQ ID: 4, SEQ ID: 5, SEQ ID: 6, SEQ ID: 7, SEQ ID: 8, SEQ ID: 9, SEQ ID: 10, SEQ ID: 11, SEQ ID: 12

BRIEF DESCRIPTION OF ABBREVIATIONS:
ACN Acetonitrile
Ac2O Acetic anhydride
Bn Benzyl
Boc t-Butyloxycarbonyl
COMU (1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate
DBU l,8-Diazabicyclo[5.4.0]undec-7-ene
DCC Dicyclohexylcarbodiimide
DCM Dichloromethane
DEPBT 3-(Diethoxyphosphoryloxy)-1, 2, 3-benzotriazin-4(3H)-one
DIC/DIPC Diisopropylcarbodiimide
DIPEA Diisopropylethylamine
DMF N,N-Dimethylformamide
DMAP 4-DimethylaminopyridineTFA Trifluoroacetic acid
EDC Ethyl-dimethylaminopropylcarbodiimide
EDT 1,2-ethanedithiol
Fmoc 9-fluorenylmethyloxycarbonyl
h Hour/s
HOBt.H2O 1-Hydroxybenzotriazole hydrate
HOAt l-Hydroxy-7-azabenzotriazole
HATU 2-(7-Aza-lH-benzotriazole-l-yl)-l,l,3,3- tetramethyluroniumhexafluorophosphate
HBTU 3-[Bis(dimethylamino)methyliumyl]-3H- benzotriazol-l-oxide hexafluorophosphate
HCTU O-(1H-6-Chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
HFIP 1,1,1,3,3,3-Hexafluoroisopropanol
HPLC High performance liquid chromatography
LPPS liquid phase peptide synthesis
MeOH Methanol
min Minutes
Oxy-B 5-(hydroxyimino)-1,3-dimethylpyrimidine-2,4,6(1H,3H,5H)-trione
Oxyma/
OxymaPure Ethyl 2-cyano-2-hydroxyimino-acetate
Pbf 2,2,4,6,7-pentamethyldihydrobenzofuran-5- sulfonyl
Pd(PPh3)4 Tetrakis(triphenylphosphine)palladium(0)
PyBOP (Benzotriazol-l-yloxy)- tripyrrolidinophosphoniumhexafluorophosphate
SPPS Solid phase peptide synthesis
tBu tert-Butyl
TBTU N,N,N’,N'-Tetramethyl-0-(benzotriazol-l- yl)uroniumtetrafluoroborate
TES Triethylsilane
TFA Trifluoroacetic acid
TFE Trifluoro ethanol
TIS Triisopropylsilane
Trt Trityl

DETAILED DESCRIPTION
As used herein Solid Phase Peptide Synthesis (“SPPS”) is the method and system that is most commonly used to synthesize polypeptides and amino acid sequences. SPPS involves coupling an activated amino acid to a solid support. The term “solid phase synthesis” (SPPS) can also be defined as a process in which a peptide anchored by its C-terminal amino acid to a resin is assembled by the sequential addition of the optionally protected amino acids constituting its sequence.
This solid support is usually a polymeric resin bead that is functionalized (such as with an NH2 group). The next amino acid (which generally has its NH2 terminus protected via a Fmoc, Boc or other terminal protecting group) is reacted with the resin such that the functionalized group on the resin reacts with and binds to the activated -COOH group of the terminal amino acid. In this manner, the terminal amino acid is covalently attached to the resin.
Then, in the next step, the NH2 terminus of the terminal amino acid is deprotected, thereby exposing its NH2 group for the next reaction. Accordingly, a new amino acid (AA) is introduced. This new amino acid has its NH2 terminus protected via a protecting group (such as an Fmoc, Boc or another protecting group). As such, when this new amino acid is added, the activated ester from the new amino acid reacts with the newly deprotected NH2 group of the terminal amino acid, thereby coupling these two amino acids together. Once this new amino acid has been coupled, it likewise has a protected NH2 group that may be subsequently deprotected and reacted with the next amino acid. By doing this repetitive, iterative process over and over, the entire amino acid sequence may be constructed. Once the entire sequence has been constructed, the sequence may be uncoupled (cleaved) from the resin and deprotected, thereby producing the amino acid sequence. The amino acids side-chain functional groups are optionally protected with groups which are generally stable during coupling steps and during a-amino protecting group removal, and which are themselves removable in suitable conditions. Such suitable conditions are generally orthogonal to the conditions in which the a-amino groups are deprotected. The protecting groups of amino acids side-chain functional groups which are used in the present disclosure are generally removable in acidic conditions, as orthogonal to the basic conditions generally used to deprotect Fmoc protecting group. Those skilled in the art will appreciate how such side chains or other group may be constructed, protected, and subsequently deprotected during the synthesis process.
The solid phase synthesis (SPPS) in present invention is carried out on a resin i.e. insoluble polymer which is acid sensitive. An acid sensitive resin is selected from a group comprising Rink amide resin (RAR), Seiber amide resin, Chlorotrityl resin (CTC), Sasrin, Wang Resin, 4-methytrityl chloride, TentaGel S, TentaGel TGA, NovaSyn TGT resin, HMPB-AM resin, 4-(2-(amino methyl)-5-methoxy)phenoxy butyric acid anchored to polymeric resin MBHA, 4-(4-(amino methyl)-3-methoxy)phenoxy butyric acid anchored to polymeric resin MBHA and 4-(2-(amino methyl)-3,3-dimethoxy)phenoxy butyric acid anchored to polymeric resin MBHA include, most preferred super acid labile resin is Rink amide resin (RAR).
Resin such as Rink amide resin, Seiber amide or CTC resin are swelled in suitable solvent such as but not limited to DCM, DMF or mixture thereof, by stirring at 10-40 °C, preferably at 25-30 °C for 0.5-1 h followed by washing with DCM, DMF or mixture thereof.
The term "amino acid" as used herein refers to an organic compound comprising at least one amino group and at least one acidic group. The amino acids may be from the group of the amino acids encoded by the genetic code and they may be natural amino acids which are not encoded by the genetic code, as well as synthetic amino acids. Commonly known natural amino acids which are not encoded by the genetic code are e.g., ?-carboxyglutamate, ornithine, phosphoserine, D-alanine and D-glutamine. Commonly known synthetic amino acids comprise amino acids manufactured by chemical synthesis, i.e. D-isomers of the amino acids encoded by the genetic code such as D-alanine and D-leucine, Aib (a-aminoisobutyric acid), Abu (a-aminobutyric acid), Tle (tert-butylglycine), ß-alanine, 3-aminomethyl benzoic acid, anthranilic acid. . Amino acid residues may be identified by their full name, their one-letter code, and/or their three-letter code.
The term “polypeptide” and “peptide” as used herein means a compound composed of at least two constituent amino acids connected by peptide bonds (amide bond) formed by removal of water from the amino acids from which they formally are build.
The term “fragment” as used herein, refers to a portion of an amino acid sequence wherein said portion is smaller than the entire amino acid sequence of Tirzepatide. The fragment can be protected with N-terminal protection and side chain protection as provided herein. Fragment also refers to peptides except Tirzepatide as provided herein.
The term "Fmoc protected amino acid" as used herein refers to amino acids with Fmoc protection on a-amino group of the amino acids. “Fmoc protected serine” refers to serine with Fmoc protection on a-amino group
As used herein the term "protecting group" refers to a labile chemical moiety which is known in the art to protect reactive groups including without limitation, hydroxyl, amino and thiol groups, against undesired reactions during synthetic procedures. Protecting groups are typically used selectively and/or orthogonally to protect sites during reactions at other reactive sites and can then be removed to leave the unprotected group as is or available for further reactions. Protecting groups as known in the art are described generally in Greene's Protective Groups in Organic Synthesis, 4th edition, John Wiley & Sons, New York, 2007.
The term "orthogonally protected" refers to functional groups which are protected with different classes of protecting groups, wherein each class of protecting group can be removed in any order and in the presence of all other classes (see, Barany et al., J. Am. Chem. Soc., 1977, 99, 7363-7365; Barany et al., J. Am. Chem. Soc., 1980, 102, 3084-3095). Orthogonal protection is widely used in for example automated oligonucleotide synthesis. A functional group is deblocked in the presence of one or more other protected functional groups which is not affected by the deblocking procedure. This deblocked functional group is reacted in some manner and at some point a further orthogonal protecting group is removed under a different set of reaction conditions. This allows for selective chemistry to arrive at a desired compound.
Examples of hydroxyl protecting groups include without limitation, acetyl, t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, bis(2-acetoxyethoxy)methyl (ACE), 2-trimethylsilylethyl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl, [(triisopropylsilyl)oxy]methyl (TOM), benzoylformate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, benzoyl, p-phenylbenzoyl, 9-fluorenylmethyl carbonate, mesylate, tosylate, triphenylmethyl (trityl), monomethoxytrityl, dimethoxytrityl (DMT), trimethoxytrityl, 1(2-fluorophenyl)-4-methoxypiperidin-4-yl (FPMP), 9-phenylxanthine-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl (MOX). Wherein more commonly used hydroxyl protecting groups include without limitation, benzyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, benzoyl, mesylate, tosylate, dimethoxytrityl (DMT), 9-phenylxanthine-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl (MOX).
Examples of amino protecting groups include without limitation, carbamate-protecting groups, such as 2-trimethylsilylethoxycarbonyl (Teoc), 1-methyl-1-(4-biphenylyl)ethoxycarbonyl (Bpoc), t-butoxycarbonyl (Boc), allyloxycarbonyl (Alloc), 9-fluorenylmethyloxycarbonyl (Fmoc), and benzyloxycarbonyl (Cbz); amide-protecting groups, such as formyl, acetyl, trihaloacetyl, benzoyl, and nitrophenylacetyl; sulfonamide-protecting groups, such as 2-nitrobenzenesulfonyl; and imine- and cyclic imide-protecting groups, such as phthalimido and dithiasuccinoyl.
Examples of thiol protecting groups include without limitation, triphenylmethyl (Trt), benzyl (Bn), and the like.
The term “N-terminal protecting group” or “N-terminal protection” or "terminal protecting group" as used herein refers to the protecting group for the a-amino group of the amino acids or of the peptides used in the preparation of Tirzepatide, or of the complete Tirzepatide sequence, which is cleaved either prior to the coupling to elongate the peptide sequence or at the end of the peptide elongation. Preferably, the N-terminal protecting group is 9-fluorenylmethyloxycarbonyl (Fmoc) or tert-butyloxycarbonyl (Boc). Deprotecting the Boc or Fmoc group from the completed-protected-coupled-product, comprises reacting the completed-protected-coupled-product with an acid in the case of Boc, or a base in the case Fmoc.
The term "deprotecting agent" as used herein refers to a reagent or reagent system (reagent(s), and solvent) useful for removing a protecting group. Deprotecting agents are acids, bases or reducing agents. For example, removal of the benzyl (Bn) group is generally accomplished by reduction (hydrogenolysis), while removal of carbamates (e.g. Boc group) is generally effected by use of acids (e.g. HC1, TFA, etc.) optionally with mild heating.
In particular, the Fmoc protecting group is cleaved by treatment with a suitable secondary amine selected from the group consisting of piperidine, pyrrolidine, piperazine and DBU, preferably piperidine. More preferably, Fmoc deprotection is carried out by using a 20% solution of piperidine in DMF. An additive such as formic acid, boric acid, citric acid can be optionally added during Fmoc deprotection to facilitate the reaction. In an aspect, after deprotection of Fmoc the resulting peptide is optionally washed with HOBt.H2O, formic acid, citric acid, and ascorbic acid in presence of solvent used for coupling, preferable washing using HOBt.H2O in DMF.
The term "side chain protection" as used herein is a protecting group for an amino acid side-chain chemical function which is not removed when the terminal protecting group is removed and is stable during coupling reactions. Preferably, side-chain protecting groups are included to protect side-chains of amino acids which are particularly reactive or labile, to avoid side reactions and/or branching of the growing molecule. Illustrative examples include acid-labile protecting groups, as for instance tert-butyloxycarbonyl (Boc), alkyl groups such as tert-butyl (tBu), trityl (Trt), 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) and the like. Other protecting groups may be efficiently used as it is apparent to the person skilled in the art.
The epsilon amino group of lysine in position 20 of Tirzepatide is protected with a protecting group (Xn) such as allyloxycarbonyl (Alloc), 4-methyltrityl (Mtt), methoxytrityl [(4-methoxyphenyl) diphenylmethyl] (Mmt), dichlorodiphenyldichloroethylene (Dde), ivDde,
In one embodiment of the invention, the coupling of amino acids may be carried out in the presence of a coupling agent and optionally in the presence of coupling additive.
The term “coupling agent” as used herein, may be selected from the group comprising of N,N'- diisopropylcarbodiimide (DIC or DIPC), N,N'-dicyclohexylcarbodiimide (DCC), (Benzotriazol-1-yloxy)tripyrrolidinophosphoniumhexafluorophosphate (PyBOP), [Ethyl cyano(hydroxyimino)acetato-O2]tri-1-pyrrolidinylphosphonium hexafluorophosphate (PyOxim), benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), N,N,N',N'-Tetramethyl-0-(benzotriazol-l-yl)uroniumtetrafluoroborate (TBTU), 2-(7-Aza-lH-benzotriazole-l-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HATU), 2-(lH-benzotriazole-l-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 2-(6-Chloro-1-H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HCTU), (1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU), ethyl-dimethylaminopropylcarbodiimide (EDC), (3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one) (DEPBT) etc,.
Coupling additive, when used in the coupling reaction, reduces loss of configuration at the carboxylic acid residue, increases coupling rates and reduces the risk of racemization. The additive may be selected from the group comprising 1- hydroxybenzotriazole.monohydrate (HOBt.H2O), 2-hydroxypyridine N-oxide, N-hydroxysuccinimide, 1-hydroxy- 7-azabenzotriazole (HOAt), endo-N-hydroxy-5-norbornene-2, 3-dicarboxamide and ethyl 2- cyano-2-hydroxyimino-acetate (OxymaPure), 5-(Hydroxyimino)l,3-dimethylpyrimidine-2,4,6-(lH,3H,5H)-trione (Oxy-B).
The term "suitable base" as used means a base that is useful for effecting the subject reaction. One of skill in the art is aware of the many bases (organic and inorganic bases) regarded as useful in the art for the purpose of the particular reaction. Suitable bases are also exemplified by the bases disclosed herein for the specific reaction. In one embodiment of the invention, the coupling reaction may be carried out in the presence of a base selected from the group of tertiary amines comprising diisopropylethylamine (DIPEA), triethylamine, collidine, N- methylmorpholine, N-methylpiperidine etc.
In one embodiment of the invention, the coupling reaction may be carried out in the presence of an inorganic base selected from the group consisting of magnesium chloride, zinc chloride or copper chloride is optionally added during coupling stage.
The term "suitable solvent" as used herein means a solvent that is useful for effecting the subject reaction. One of skill in the art is aware of the many solvents regarded as useful in the art for the purpose of the particular reaction. Suitable solvents are also exemplified by the solvents disclosed herein for the specific reaction.
In one embodiment of the invention, the coupling reaction, either involving peptides or amino acids, takes place in the presence of a solvent selected from the group comprising dimethylformamide (DMF), dimethylacetamide (DMAc), dimethylsulfoxide (DMSO), dichloromethane (DCM), chloroform (CHCl3), tetrahydrofuran (THF), 2-methyl tetrahydrofuran (2Me-THF) and N-methyl pyrrolidine (NMP).
The preferred coupling agent, coupling additive and base used in present invention in any of the above process is DIC/HOBt.H2O; DIC/OxymaPure; HBTU/HOBt.H2O/DIPEA, HBTU/OxymaPure/DIPEA; HBTU/Oxy-B/DIPEA; HATU/HOAt/DIPEA or PyBOP/ HOBt.H2O/DIPEA.
In a preferred embodiment, Gly and C20-monoester are coupled using DIC/OxymaPure. In another preferred embodiment, Trp, Gln, Phe, Ile, AEEA, ?-Glu, Ala, Val, Pro and Leu are coupled using HBTU/DIPEA/ HOBt.H2O. In another preferred embodiment, Ser, Thr, Lys, Aib, Glu, Tyr, and Asp are coupled using HATU/DIPEA/OxymaPure
The amount of individual coupling agents used may range from about 1 to about 6 molar equivalents, per molar equivalent of resin with respect to resin loading capacity. Preferably, 3 molar equivalents of individual coupling agents per molar equivalent of the resin with respect to resin loading capacity may be used.
The solvent used in said coupling reaction is selected from DMF, DCM, NMP or DMSO in combination between them; preferably DMF.
The coupling temperature is usually in the range of from 10 to 40 °C; preferably 25 to 30 °C.
Additionally, the unreacted sites on the resin are optionally capped, to avoid truncated sequences and to prevent any side reactions, by a short treatment with a large excess of a highly reactive unhindered reagent, which is chosen according to the unreacted sites to be capped, and according to well-known peptide synthesis techniques. For example, capping may be performed using acetic anhydride in presence of base such as DIPEA, TEA, Pyridine and like. The aim of capping is preventing the occurrence of product like or closely eluting impurities (i.e. products lacking an amino acid building block at one position), which are probably hard to separate from the desired final product.
Deprotection and cleavage conditions generally depend on the nature of the protecting groups and of the resin used. The concomitant removal of amino acid side chain protection and polymer support of the peptide from the resin involves treating the protected peptide anchored to the resin with a cleaving agent that comprises an acid and at least one scavenger. The peptide cleavage reagent (or cleaving agent) used in the process of the present invention is a cocktail mixture of acid, scavengers and solvents. The acidic is preferably based on an acidic material such as TFA, and contains scavenger reagents including, but not limited to, water, triethylsilane (TES), DTT, triisopropylsilane (TIS/TIPS), 2, 2’-(ethylenedioxy)diethane, acetyl cystein, DMS, phenol, and cresol or mixture thereof and water; preferably TFA: TIS: phenol: water. The relative ratio of acidic material to scavenger to water may be from about 70 % to about 90% acidic material, from about 10 % to about 30% scavenger. Preferably the ratio of TFA: TIPS: phenol: water is 80 %: 5 %: 10%: 5 %.
In one embodiment of the invention, acid such as formic acid, boric acid, citric acid is optionally added along with piperidine during deprotection of Fmoc protecting group.
Orthogonal deprotection of Lys20 is carried out using hydrazine hydrate when orthogonal protecting group is Dde. When orthogonal protecting group is Mtt, deprotection is carried out using HFIP, TFE, TES in MDC. When orthogonal protecting group is Alloc, deprotection is carried out using Pd(PPh3)4 and phenylsilane.
The deprotection temperature is usually in the range of from 10 to 40 °C; preferably 25 to 30 °C in suitable solvent such as but not limited to DMF, DCM. The epsilon amino group of lysine in position 20 is coupled with N-terminal and side chain protected or un-protected eicosanedioic acid-?-Glu-AEEA-AEEA-OH or by employing additional coupling/deprotection cycles as to extend the Lys20 side-chain involving Fmoc-AEEA-OH, Fmoc-Glu(OH)-OtBu and HOOC—(CH2)18-COOtBu to obtain protected Tirzepatide- peptidyl resin. The coupling is performed using suitable coupling agent such as but not limited to DIC, HATU, HBTU, PyBOP, PyOxim, BOP, HCTU, COMU, and DEPBT in presence of additive such as but not limited to HOBt.H2O, OxymaPure and base such as but not limited to DIPEA, colliding base in suitable solvent such as but not limited to DMF and stirring at 10-40°C, preferably at 25-30 °C for 2 to 6 h followed by washing with solvent such as but not limited to DCM, DMF and IPA or mixture thereof.
In a preferred embodiment, when SPPS is used, the protected Tirzepatide sequence is finally deprotected and cleaved from the resin, either simultaneously or in two steps, providing crude Tirzepatide, which may optionally be purified.
Concomitant cleavage from the resin and side chain protecting group removal is carried out by treating the peptide with a cleaving agent comprising a mixture of TFA, TIPS, Phenol, H2O at 10-40 °C, preferably at 25-30 °C for 2 to 4 h.
The crude Tirzepatide obtained may be isolated by precipitation using suitable solvents such as but not limited to MTBE, toluene, diisopropyl ether, hexane, heptane, isopropyl ether optionally purified by crystallization or chromatographic techniques well known in the art.
In an embodiment, purification involves reverse phase chromatographic (RP-HPLC) purification. In another embodiment the RP-HPLC employs mobile phase selected from group comprising of Trifluoroacetic acid, formic acid, citric acid, Ammonium bicarbonate, Ammonium carbonate, trisaminomethane (TRIS) buffer, acetonitrile, 2-propanol, water. In an embodiment crude tirzepatide is subjected to one or more RP-HPLC purification.
In another embodimernt, pure tirzepatide is isolated by subjecting the fractions collected from RP-HPLC purification to distillation, treatment with NaOH solution and followed by lyophilization.
The inventors of the present process have found that the use of the above described polypeptide fragments in preparation of Tirzepatide or its pharmaceutically acceptable salt, provides Tirzepatide in better yield and high purity, which makes it suitable for large scale industrial production
The term "pure Tirzepatide" refers to Tirzepatide which have purity over 99%, preferably over 99.5%, more preferably over 99.9%.
The terms "about" as used herein refers to as modifying a term or value such that it is not an absolute. Such terms will be defined by the circumstances and the terms that they modify as those terms are understood by those of skill in the art. This includes, at very least, the degree of expected experimental error, technique error and instrument error for a given technique used to measure a value. The term "about" when used in the present application preceding a number and referring to it, is meant to designate any value which lies within the range of ±10 %, preferably within a range of ±5 %.
The terms "comprising" and "comprises" as used herein are to be construed as open ended and mean the elements recited, or their equivalents in structure or function, plus any other element or elements which are not recited.
As used herein, when an amino acid abbreviation appears with a number above the amino acid, the number refers to the corresponding amino acid position in the final Tirzepatide product. The numbers are provided for convenience and the appearance or absence of such numbers in a sequence does not influence the amino acid sequence or the peptide indicated in such sequence.
First aspect of the present invention relates to an improved process of preparation of Tirzepatide comprising use of Fmoc-Ser39-NH2. Structure of Fmoc-Ser39-NH2 is depicted below:

In an embodiment of the invention, in step (a) of first aspect Fmoc-Ser39-NH2 is attached to a solid support via its side chain; by solid-phase synthesis to obtain Fmoc-Ser39(resin)-NH2.
In an embodiment, Fmoc-Ser39(resin)-NH2 obtained in step (a) of first aspect is specifically Fmoc-Ser39 (CTC)-NH2 as depicted below:

The solid support in step (a) of first aspect may be any support known in the art that is suitable for use in solid-phase peptide synthesis. Such supports are able to react, commonly via a linking group, with a side chain of an amino acid, more preferably a nucleophilic amino acid side chain to form a bond which is stable during the acylation and deprotection cycles, yet allow release of the peptide amide from the resin during concomitant cleavage.
The solid phase synthesis (SPPS) in present invention is carried out on a resin selected from 2-chlorotrityl chloride resin or 4-methoxytrityl polystyrene.
In one embodiment of first aspect, in step (c) side chain deprotection on lysine from fragment obtained in step (b) is carried out using1-5% hydrazine hydrate in DMF, wherein Xn is Dde. Wherein Xn is Mtt, deprotection is carried out using HFIP, TFE, TES in MDC. Where in Xn is Alloc, deprotection is carried out using Pd(PPh3)4 and phenylsilane.
In an embodiment of first aspect, conversion of SEQ ID: 2 to Tirzepatide comprises reacting with side chain protected or un-protected fragment eicosanedioic acid-?-Glu-AEEA-AEEA-OH OR sequentially reacting the resin with AEEA, AEEA, ?-glutamic acid(OtBu), eicosanedioic acid(OtBu) OR sequentially reacting the resin with AEEA-AEEA, ?-glutamic acid(OtBu), eicosanedioic acid(OtBu) to obtain SEQ ID: 3 followed by concomitant cleavage of solid support and protecting group of ensuing peptide using a cleaving agent.
In one embodiment, SEQ ID: 3 is converted to Tirzepatide by concomitant cleavage of solid support and protecting group of ensuing peptide using a cleaving agent.
In one embodiment, eicosanedioic acid(OtBu) is also referred as C20-diacid(OtBu), C20-monoester or 20-(tert-butoxy)-20-oxoicosanoic acid and eicosanedioic acid is also referred as C20-diacid or C20-acid. In one embodiment fragment eicosanedioic acid-?-Glu-AEEA-AEEA-OH specifically is C20-monoester-?-Glu(OtBu)-AEEA-AEEA-OH and represented as SC-1 by following structure as:

Figure 1: (Preparation of Tirzepatide according to first aspect)

Second aspect of the present invention relates to an improved process of preparation of Tirzepatide comprising using SEQ ID: 4 and SEQ ID: 5;
In an embodiment SEQ ID: 4 used in step (c) of second aspect is Fmoc-Phe6-Thr7[Psi(Me, Me)Pro]-OH wherein Fmoc-Phe6-Thr7[Psi(Me, Me)Pro]-OH is a pseudoproline dipeptide and indicates that the Thr is pseudoproline protected, according to the following formula:

In an embodiment SEQ ID: 5 used in step (d) of second aspect is Fmoc-Gly4-Thr5[Psi(Me, Me)Pro]-OH wherein Fmoc-Gly4-Thr5[Psi(Me, Me)Pro]-OH is a pseudoproline dipeptide and indicates that the Thr is pseudoproline protected, according to the following formula:

In one embodiment, in step (f) of second aspect side chain deprotection on lysine from fragment obtained in step (e) is carried out in similar manner as described herein above.
In an embodiment, conversion of SEQ ID: 8 to Tirzepatide according to second aspect comprises reacting SEQ ID: 8 obtained after deprotection of protecting group (Xn) from Lys20 obtained in step-f of second aspect, with side chain protected or un-protected fragment eicosanedioic acid-?-Glu-AEEA-AEEA-OH OR sequentially reacting the resin with AEEA, AEEA, ?-glutamic acid(OtBu), eicosanedioic acid(OtBu) OR sequentially reacting the resin with AEEA-AEEA, ?-glutamic acid(OtBu), eicosanedioic acid(OtBu) as described in first aspect to obtain SEQ ID: 9 followed by concomitant cleavage of solid support and protecting group of ensuing peptide using a cleaving agent.
In one embodiment, SEQ ID: 9 is converted to Tirzepatide by concomitant cleavage of solid support and protecting group of ensuing peptide using a cleaving agent as described hereinabove for first aspect of this invention.
In one embodiment Tirzepatide is prepared by following procedure as depicted in Scheme 2.
Scheme-2: (Preparation of Tirzepatide according to second aspect)

Third aspect of the present invention relates to an improved process of preparation of Tirzepatide comprising:
a. loading of Fmoc protected serine to a resin;
b. sequential coupling of amino acids based on the sequence of peptide backbone of Tirzepatide till Phe6 in the presence of coupling agent
c. coupling of Fmoc-Aib2-Glu3-Gly4-Thr5-OH (SEQ ID: 10) in the presence of coupling agent
d. coupling of Tyr1 OH with in the presence of coupling agent
e. deprotection of protecting group (Xn) from Lys20 and converting the resulting peptide to Tirzepatide.
In one embodiment Tirzepatide is prepared by following procedure as depicted in Scheme 3.
Scheme 3: (Preparation of Tirzepatide according to Third aspect)

Fourth aspect of the present invention relates to an improved process of preparation of Tirzepatide comprising;
a. loading of Fmoc protected serine to a resin;
b. sequential coupling of amino acids based on the sequence of peptide backbone of Tirzepatide till Gln19 in the presence of coupling agent
c. coupling of Tyr1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Ile12-Aib13-Leu14-Asp15-Lys16-Ile17-Ala18-OH (SEQ ID: 11) in the presence of coupling agent
d. deprotection of protecting group (Xn) from Lys20 and converting the resulting peptide to Tirzepatide.
.
Scheme 4: (Preparation of Tirzepatide according to Fourth aspect)



Fifth aspect of the present invention relates to an improved process of preparation of Tirzepatide comprising;
a. loading of Fmoc protected serine to a resin;
b. sequential coupling of amino acids based on the sequence of peptide backbone of Tirzepatide till Gln19 in the presence of coupling agent
c. coupling of Lys16-Ile17-Ala18-OH (SEQ ID: 12) in the presence of coupling agent
d. sequential coupling of amino acids based on the sequence of peptide backbone of Tirzepatide till Tyr1 in the presence of coupling agent
a. deprotection of protecting group (Xn) from Lys20 and converting the resulting peptide to Tirzepatide.

Scheme 5: (Preparation of Tirzepatide according to Fifth aspect)

In one embodiment of the invention, deprotecting agents for Fmoc deprotection is base in suitable organic solvent. The base used may be an inorganic or organic base. Preferably the base is an organic base selected from the group comprising piperidine, pyrrolidine, piperazine, tert-butylamine, DBU and diethylamine, preferably piperidine in DMF.
In one embodiment of the invention, additive for Fmoc deprotection is selected from the group comprising formic acid, citric acid and boric acid or mixture thereof.
In one embodiment of the invention, Fmoc deprotection is carried out with deprotecting agents with additive wherein Tirzepatide is synthesized by solid phase peptide synthesis (SPPS) or liquid phase peptide synthesis (LPPS). Further, approach to solid phase synthesis can be liner synthesis or fragment synthesis.
In one embodiment, the process of the present invention comprises preparing peptides using SPPS, LPPS (also referred as solution phase synthesis) or a hybrid SPPS/LPPS approach
In an embodiment the fragments provided herein can be prepared by solution phase synthesis
In solution phase synthesis, a reaction or process is carried out in solution rather than with the use of a solid support such as employed in conventional solid phase peptide syntheses. Solution phase synthesis is analogous to SPPS, except the growing peptide is not bound to the resin and the C-terminus of the peptide is either a nonreactive amide or a protected ester. In solution phase synthesis, coupling is performed in suitable solvent, and usually in the presence of coupling agent and suitable base. The side chain protecting groups, which are present during solid phase synthesis, are commonly retained during solution phase coupling to ensure the specific reactivity of the terminal ends of the fragments. These side chain protecting groups are typically not removed until desired peptide has been formed.
The invention is further exemplified by the following non-limiting examples, which are illustrative representing the preferred modes of carrying out the invention. The invention's scope is not limited to these specific embodiments only but should be read in conjunction with what is disclosed anywhere else in the specification together with those information and knowledge which are within the general understanding of the person skilled in the art.

Examples:
Example 1: Synthesis of Tirzepatide using Fmoc-Ser(OH)-NH2 [Aspect 1, scheme 1]
2-CTC (100 g, 0.8 mmol/g substitution) was washed with DCM in a reaction flask, solvent removed using vacuum and allowed to swell in DCM. Fmoc-Ser(OH)-NH2 (2.0 eq), and DIPEA in DMF was added to the resin made according the above procedure, stirred for 2-3 h. After the reaction complies, the resin is washed with DMF. Substitution obtained after first amino acid loading was 0.4 mmol/g. Capping was performed using 10 % methanol in MDC and DIPEA and resin was washed with DMF.
After 1st amino acid attachment, Fmoc deprotection was performed using 5- 15 % piperidine in DMF with 1 % boric acid mixture and reaction was monitored using Kaiser test. After the reaction complies, the resin is washed with DMF/0.5% formic acid followed by DMF washings were monitored by Chloranil test.
The rest of the amino acids were coupled as per the sequence using following coupling conditions:
The rest of the amino acids were coupled as per the sequence. Fmoc-AA-OH/Boc-AA-OH, coupling agent and additive were dissolved in DMF and the reaction mixture was added to the resin, stirred for 1.5 – 2.0 h and reaction monitored using Kaiser test. After reaction complies resin was washed with DMF to obtain SEQ ID: 1.
Fmoc-Axx-OH/ Boc-Axx-OH/Fragment, coupling agent, additive details given below:
i) Gly and C20-monoester are coupled using DIC/OxymaPure
ii) Trp, Gln, Phe, Ile, AEEA, ?-Glu, Ala, Val, Pro and Leu are coupled using HBTU/DIPEA/ HOBt.H2O
iii) Ser, Thr, Lys, Aib, Glu, Tyr, and Asp are coupled using HATU/DIPEA/OxymaPure
All Fmoc-deprotections were performed by using 5- 15 % piperidine in DMF with 0.5 % boric acid. After completion of linear synthesis, Mtt protecting group on Lys was removed using a mixture of HFIP, TES, TFE in DCM and and stirred at 25-30°C till completion of reaction. After the reaction complies, solvent was removed using vacuum and resin was washed with DCM wash followed by DMF in 1 % DIPEA and fresh DMF to obtain SEQ ID: 2.
Sequential coupling of Fmoc-AEEA-OH, Fmoc-AEEA-OH and Fmoc-Glu-OtBu and 20-(tert-Butoxy)-20-oxoicosanoic acid was done using HBTU/ HOBt.H2O/ DIPEA in DMF for 2-3 h and reaction was monitored using Kaiser test. After reaction complies resin was washed with DMF to obtain Boc-Tyr1(tBu)-Aib2-Glu3(OtBu)-Gly4-Thr5(tBu)-Phe6-Thr7(tBu)-Ser8(tBu)-Asp9(OtBu)-Tyr10(tBu)-Ser11(tBu)-Ile12-Aib13-Leu14-Asp15(OtBu)-Lys16(Boc)-Ile17-Ala18-Gln19(Trt)-Lys20(AEEA-AEEA-?-Glu(OtBu)-C20-diacid(OtBu)-Ala21-Phe22-Val23-Gln24(Trt)-Trp25(Boc)-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser32(tBu)-Ser33(tBu)-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39(Resin)-NH2 (SEQ ID: 3)
Resin was washed with DMF followed by DCM, MeOH and MTBE and dried. Cleavage cocktail mixture of TFA/TIPS/Phenol/H2O (80: 5: 10: 5 %) was added to the resin and stirred for 3-4 h. After reaction complies, the reaction mixture was filtered, filtrate was evaporated and crude peptide was isolated using chilled MTBE. Crude peptide was washed with MTBE and dried to obtain crude Tirzepatide. Crude: 180 g and product content 54.5 g.
The crude peptide was purified by two cycles RP-HPLC. In first cycle, crude Tirzepatide was dissolved in 8 volumes of 10 % acetonitrile in water containing acidic buffer in water. DAC (stationary phase C8-10 µ-100 Å) was washed with 90:10 (acetonitrile: water containing acidic buffer) for two column volumes followed by equilibrating with 100% mobile phase A [90% (acidic buffer in water) and 10% ACN] for two column volumes. Filtered the above crude solution and injected into the column.
Mobile phase A: [90 % acidic buffer in water and 10 % acetonitrile] at pH 2.0 to 4.0. Mobile Phase B: 100% acetonitrile. Wavelength: 214 nm.
Gradient method containing mobile phase B from 10 % to 40 % over a period 140 minutes, in which, product elutes at 25 % to 30 % near about 90 – 110 minutes was performed. Above 90 % pure fractions were pooled for RP-2. The column was regenerated by washing with 90:10 (acetonitrile: water containing acidic buffer in water) for two column volumes and re equilibrated with 100 % mobile phase A [90 % acidic buffer in water and 10 % ACN] for two column volumes prior to next injection sequence. The above procedure was followed until the completion of the crude. Fractions having purity more than 50.0 % were pooled and reinjected to obtain fractions having purity above 90 %, by following the same procedure for elution mentioned in the above. All main fractions having purity more than 90 % were pooled and subjected to stationary phase C8-10 µ-100 Å for further purification.
Mobile phase A: [90 % 10 mM Ammonium bicarbonate in water and 10 % acetonitrile] at pH 7.5 to 8.0. Mobile Phase B: 100% acetonitrile. Wavelength: 214 nm.
Gradient method containing mobile phase B from 10 % to 35 % over a period 140 minutes, in which, product elutes at 15 % to 25 % near about 90 – 100 minutes was performed. Fractions above 99% purity were pooled. The column was regenerated by washing with 90:10 (acetonitrile: water containing 10 mm ammonium bicarbonate in water) for two column volumes and re equilibrated with 100 % mobile phase A [95 % 10 mM ammonium bicarbonate in water and 5 % acetonitrile] for two column volumes prior to next injection sequence. Fractions having purity more than 60.0 % were pooled and reinjected to obtain fractions having purity above 99.0% by following the same procedure for elution mentioned in the above. All pooled passing fractions were concentrated and lyophilized to get pure Tirzepatide API. Yield: 18.95 g.

Example 2: Synthesis of SEQ ID: 11 [Aspect 4, scheme 4]
Fmoc-Ala-OH was loaded to 2-CTC resin (100 g) to achieve substitution of 1.0 mmol/g in DIPEA/DCM. All the amino acids were coupled sequentially to the resin with Fmoc-/tBu strategy.
The rest of the amino acids were coupled as per the sequence using following coupling conditions:
Fmoc-AA-OH/Boc-AA-OH, coupling agent and additive were dissolved in DMF and the reaction mixture was added to the resin, stirred for 1.5 – 2.0 h and reaction monitored using Kaiser test. After reaction complies resin was washed with DMF.
Fmoc-AA-OH/ Boc-AA-OH, coupling agent, additive details given below:
i) Gly, Leu, and Ile are coupled using DIC/OxymaPure
ii) Tyr, Aib, Glu, Thr, Phe, Thr, Ser, Asp, and Lys are coupled using HBTU / DIPEA / OxymaPure
iii) All Fmoc-deprotection were performed by using 5- 15 % piperidine in DMF with 0.5 % boric acid
Peptide was cleaved using mixture of 1% TFA/DCM and precipitated with MTBE to get crude peptide (323.4 g) and crystallized using EtOAc/ACN/n-hexane and obtained 242.5 g product.

Example 3: Synthesis of Tirzepatide using SEQ ID: 11 [Aspect 4, scheme 4]
Rink amide resin (100 g, 0.50 mmol/g) was washed with DMF in a reaction flask, solvent removed using vacuum and allowed to swell in DMF. Fmoc deprotection was done using 5% piperidine in DMF and reaction was monitored using Kaiser test. After the reaction complies, the resin is washed with DMF/0.5% formic acid followed by DMF. Fmoc-Ser(tBu)-OH (0.8 eq), HBTU, OxymaPure and DIPEA in DMF was added to the resin made according the above procedure, stirred for 2-3 h and the reaction is monitored by Kaiser test. After the reaction complies, the resin is washed with DMF followed by DCM. Substitution obtained after first amino acid loading was 0.35 mmol/g. Capping was performed using acetic anhydride and DIPEA in DMF and resin was washed with DMF.
After 1st amino acid attachment, Fmoc deprotection was performed using 5- 15 % piperidine in DMF with 0.5 % boric acid mixture and reaction was monitored using Kaiser test. After the reaction complies, the resin is washed with DMF/0.5% formic acid followed by DMF washings were monitored by Chloranil test.
The rest of the amino acids were coupled as per the sequence using following coupling conditions:
Fmoc-AA-OH/Boc-AA-OH, coupling agent and additive were dissolved in DMF and the reaction mixture was added to the resin, stirred for 1.5 – 2.0 h and reaction monitored using Kaiser test. After reaction complies resin was washed with DMF.
Fmoc-Axx-OH/ Boc-Axx-OH/Fragment, coupling agent, additive details given below:
i) Gly, Ala, Val, Pro, and Leu are coupled using DIC/OxymaPure:
ii) Trp, Gln, Phe, Ile, Ser, AEEA, ?-Glu and C20-monoester are coupled using HBTU/DIPEA/OxymaPure
iii) Lys and SEQ ID: 11 are coupled using HATU/DIPEA/OxymaPure
All Fmoc-deprotections were performed by using 5- 15 % piperidine in DMF with 0.5 % boric acid. SEQ ID: 1 was synthesized using 2-CTC resin by standard SPPS conditions and coupled to the growing peptide sequence on Rink amide resin. After completion of the fragment coupling, Alloc protecting group was removed using Pd(PPh3)4 and phenylsilane and stirred for 3-4 h under nitrogen atmosphere. After the reaction complies, solvent was removed using vacuum and resin was washed with DCM wash followed by DMF in 1 % DIPEA and fresh DMF.
Sequential coupling of Fmoc-AEEA-OH, Fmoc-AEEA-OH and Fmoc-Glu-OtBu and 20-(tert-butoxy)-20-oxoicosanoic acid was done using HBTU/ OxymaPure/ DIPEA in DMF for 2-3 h and reaction was monitored using Kaiser test. After reaction complies resin was washed with DMF to obtain Boc-Tyr1(tBu)-Aib2-Glu3(OtBu)-Gly4-Thr5(tBu)-Phe6-Thr7(tBu)-Ser8(tBu)-Asp9(OtBu)-Tyr10(tBu)-Ser11(tBu)-Ile12-Aib13-Leu14-Asp15(OtBu)-Lys16(Boc)-Ile17-Ala18-Gln19(Trt)-Lys20(AEEA-AEEA-?-Glu(OtBu)-C20-diacid(OtBu))-Ala21-Phe22-Val23-Gln24(Trt)-Trp25(Boc)-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser32(tBu)-Ser33(tBu)-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39(tBu)-Rink amide resin.
Resin was washed with DMF followed by DCM, MeOH and MTBE and dried. Cleavage cocktail mixture of TFA/TIPS/Phenol (80: 12.5: 7.5: ) was added to the resin and stirred for 3-4 h. After reaction complies, the reaction mixture was filtered, filtrate was evaporated and crude peptide was isolated using chilled MTBE. Crude peptide was washed with MTBE and dried to obtain crude Tirzepatide. Crude: 173 g and product content 50.5 g.
The crude peptide was purified by four cycles RP-HPLC.
In first cycle, crude Tirzepatide was dissolved in 8 volumes of 10 % acetonitrile in water containing acidic buffer in water. Crude Tirzepatide was dissolved in 0.2 % Trifluoroacetic acid and Acetonitrile. Octylsilane stationary phase silica was equilibrated using 0.2 % Trifluoroacetic acid and Acetonitrile. Filtered the above crude solution and injected into the column. Below mentioned mobile phases were used either alone or in combination to purify the crude Tirzepatide.
Mobile phases:
Mobile phase A Trifluoroacetic acid
(0.05 % to 0.2 %) Trifluoroacetic acid
(0.05 % to 0.2 %) Trifluoroacetic acid
(0.05 % to 0.2 %)
Mobile phase B Acetonitrile 0.1 % Trifluoroacetic acid in Acetonitrile 2-propanol: Acetonitrile (7:3)
Gradient 38 to 43 % of B in about 8 column volumes

Mobile phase A Formic acid
(0.05 % to 0.5 %) Formic acid
(0.05 % to 0.5 %) Formic acid
(0.05 % to 0.5 %)
Mobile phase B Acetonitrile 2-propanol: Acetonitrile (3:7) 2-propanol: Acetonitrile (7:3)
Gradient 35 to 40 % of B in about 8 column volumes

Mobile phase A Citric acid
(0.01 M to 0.10 M) Citric acid
(0.01 M to 0.10 M) Citric acid
(0.01 M to 0.10 M)
Mobile phase B Acetonitrile 2-propanol: Acetonitrile (1:1) 2-propanol: Acetonitrile (7:3)
Gradient 35 to 40 % of B in about 8 column volumes

Gradient elution was performed by gradually increasing the percentage of organic mobile phase in the column Column was washed with higher organic mobile phase percentage, usually of about 70 % to 90 % to remove any strongly bound compounds. All main fractions having defined purity were collected and subjected to Octylsilane stationary phase silica for further purification.
In second cycle, pure fractions solution of step 1 purification was diluted using equal amount of water and is loaded to the column. Same procedure as followed for first cycle was repeated to obtain pure fractions using below mentioned either alone or in combination.
Mobile phases:
Mobile phase A Formic acid
(0.05 % to 0.5 %) Formic acid
(0.05 % to 0.5 %) Formic acid
(0.05 % to 0.5 %)
Mobile phase B Acetonitrile 2-propanol: Acetonitrile (3:7) 2-propanol: Acetonitrile (7:3)
Gradient 35 to 40 % of B in about 8 column volumes

Mobile phase A Citric acid
(0.01 M to 0.10 M) Citric acid
(0.01 M to 0.10 M) Citric acid
(0.01 M to 0.10 M)
Mobile phase B Acetonitrile 2-propanol: Acetonitrile (1:1) 2-propanol: Acetonitrile (7:3)
Gradient 35 to 40 % of B in about 8 column volumes

In third cycle, pure fractions solution of step 2 purification was diluted using equal amount of water and is loaded to the column. Same procedure as followed for first cycle was repeated to obtain pure fractions using below mentioned mobile phase.
Mobile phase:
Mobile phase A Ammonium bicarbonate
(0.01 M to 0.10 M) TRIS buffer
(0.01 M to 0.10 M)
Mobile phase B Acetonitrile Acetonitrile
Gradient 28 to 33 % of B in about 8 column volumes

In forth cycle, pure fractions solution of step 3 purification was diluted using equal amount of water and is loaded to the column and subjected to desalting. Same procedure as followed for first cycle was repeated to obtain pure fractions using below mentioned mobile phase.
Mobile phase:
Mobile phase A Ammonium bicarbonate
(0.01 M to 0.10 M) Ammonium carbonate
(0.01 M to 0.10 M)
Mobile phase B Acetonitrile Acetonitrile
Elution Isocratic

After fourth cycle, pooled fractions are collected and subjected to distillation under vacuum at 20 °C to 40 °C to remove organic solvent and provide concentrated solution. Dilute NaOH (0.02 M to 0.10 M) is added to concentrated solution, filtered using 0.45 µm and 0.2 µm filters. The filtered solution was by freezed at about -30°C to -40°C, applied vaccum and the temperature was gradually raised to -10 oC, +5 oC, +15 oC, +30oC. to get pure Tirzepatide API. Yield: 21.81 g.
Example 4: Synthesis of SEQ ID: 12 [Aspect 5, scheme 5]
Fmoc-Ala-OH was loaded to 2-CTC resin (120 g) to achieve substitution of 1.0 mmol/g in DIPEA/DCM. All the amino acids were coupled sequentially to the resin with Fmoc-/tBu strategy.
The rest of the amino acids were coupled as per the sequence using following coupling conditions:
Fmoc-AA-OH/Boc-AA-OH, coupling agent and additive were dissolved in DMF and the reaction mixture was added to the resin, stirred for 1.5 – 2.0 h and reaction monitored using Kaiser test. After reaction complies resin was washed with DMF.
Fmoc-AA-OH/ Boc-AA-OH, coupling agent, additive details given below:
i) Ile coupled using DIC/OxymaPure
ii) Lys coupled using HBTU/DIPEA/OxymaPure
iii) Fmoc-deprotection were performed by using 5- 15 % piperidine in DMF with 0.5 % boric acid
Peptide was cleaved using mixture of 1% TFA/DCM and precipitated with MTBE to get crude peptide (93.8 g) and crystallized using EtOAc/ACN/n-hexane and obtained 74.3 g product.

Example 5: Synthesis of Tirzepatide using SEQ ID: 12 [Aspect 5, scheme 5]
Rink amide resin (100 g, 0.50 mmol/g) was washed with DMF in a reaction flask, solvent removed using vacuum and allowed to swell in DMF. Fmoc deprotection was done using 5% piperidine in DMF and reaction was monitored using Kaiser test. After the reaction complies, the resin is washed with DMF/0.5% formic acid followed by DMF. Fmoc-Ser(tBu)-OH (0.8 eq), HBTU, OxymaPure and DIPEA in DMF was added to the resin made according the above procedure, stirred for 2-3 h and the reaction is monitored by Kaiser test. After the reaction complies, the resin is washed with DMF followed by DCM. Substitution obtained after first amino acid loading was 0.35 mmol/g. Capping was performed using acetic anhydride and DIPEA in DMF and resin was washed with DMF.
After 1st amino acid attachment, Fmoc deprotection was performed using 5- 15 % piperidine in DMF with 0.5 % boric acid mixture and reaction was monitored using Kaiser test. After the reaction complies, the resin is washed with DMF/0.5% formic acid followed by DMF washings were monitored by Chloranil test.
Synthesis was continued up to Gln19 using using following coupling conditions:
Fmoc-AA-OH/Boc-AA-OH, coupling agent and additive were dissolved in DMF and the reaction mixture was added to the resin, stirred for 1.5 – 2.0 h and reaction monitored using Kaiser test.
Fmoc-AA-OH/fragment, coupling agent, additive details given below:
i) Gly, Ala, Val, Pro and Leu are coupled using DIC/OxymaPure
ii) Trp, Gln, Phe, Ile, AEEA, ?-Glu and C20-monoester are coupled using HBTU/DIPEA/OxymaPure
iii) Ser, Thr, Aib, Tyr, Glu, Asp, and SEQ ID: 12 are coupled using HATU/DIPEA/OxymaPure
All Fmoc-deprotections were performed by using 5- 15 % piperidine in DMF with 0.5 % boric acid. SEQ ID: 2 was synthesized using 2-CTC resin by standard SPPS procedure and coupled growing peptide on Rink amide resin. After SEQ ID: 2 attachment, sequential assembly was continued till Tyr1 to obtain Boc-Tyr1(tBu)-Aib2-Glu3(OtBu)-Gly4-Thr5(tBu)-Phe6-Thr7(tBu)-Ser8(tBu)-Asp9(OtBu)-Tyr10(tBu)-Ser11(tBu)-Ile12-Aib13-Leu14-Asp15(OtBu)-Lys16(Boc)-Ile17-Ala18-Gln19(Trt)-Lys(Dde)20-Ala21-Phe22-Val23-Gln24(Trt)-Trp25(Boc)-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser32(tBu)-Ser33(tBu)-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39(tBu)-Rink amide resin.
After completion of the fragment coupling, Dde protecting group was removed using hydrazine hydrate in DMF added and stirred at 25-30°C till completion of reaction. After the reaction complies, solvent was removed using vacuum and resin was washed with DCM wash followed by DMF in 1 % DIPEA and fresh DMF.
Sequential coupling of Fmoc-AEEA-OH, Fmoc-AEEA-OH and Fmoc-Glu-OtBu and 20-(tert-Butoxy)-20-oxoicosanoic acid was done using HBTU/ OxymaPure/ DIPEA in DMF for 2-3 h and reaction was monitored using Kaiser test. After reaction complies resin was washed with DMF to obtain Boc-Tyr1(tBu)-Aib2-Glu3(OtBu)-Gly4-Thr5(tBu)-Phe6-Thr7(tBu)-Ser8(tBu)-Asp9(OtBu)-Tyr10(tBu)-Ser11(tBu)-Ile12-Aib13-Leu14-Asp15(OtBu)-Lys16(Boc)-Ile17-Ala18-Gln19(Trt)-Lys20(AEEA-AEEA-?-Glu(OtBu)-C20-diacid(OtBu))-Ala21-Phe22-Val23-Gln24(Trt)-Trp25(Boc)-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser32(tBu)-Ser33(tBu)-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39(tBu)--Rink amide resin.
Resin was washed with DMF followed by DCM, MeOH and MTBE and dried. Cleavage cocktail mixture of TFA/TIPS/Phenol/H2O (80: 5: 10: 5 %) was added to the resin and stirred for 3-4 h. After reaction complies, the reaction mixture was filtered, filtrate was evaporated and crude peptide was isolated using chilled MTBE. Crude peptide was washed with MTBE and dried to obtain crude Tirzepatide. Crude: 165 g and product content 52.5 g.
The crude peptide was purified in a manner analogous to that described in example-1 to get pure Tirzepatide API. Yield: 22.5 g.

Example 6: Synthesis of Tirzepatide using sequential strategy [Sequential synthesis]
Rink amide resin (100 g, 0.50 mmol/g) was washed with DMF in a reaction flask, solvent removed using vacuum and allowed to swell in DMF. Fmoc deprotection was done using 5% piperidine in DMF and reaction was monitored using Kaiser or ninhydrin test. After the reaction complies, the resin is washed with DMF/0.5% formic acid/0.1 M HOBt.H2O followed by DMF. Fmoc-Ser(tBu)-OH (0.8 eq), HBTU, HOBt.H2O and DIPEA in DMF was added to the resin made according the above procedure, stirred for 2-3 h and the reaction is monitored by Kaiser or ninhydrin test. After the reaction complies, the resin is washed with DMF followed by DCM. Substitution obtained after first amino acid loading was 0.35 mmol/g. Capping was performed using acetic anhydride and DIPEA in DMF and resin was washed with DMF.
After 1st amino acid attachment, Fmoc deprotection was performed using 5- 20 % piperidine in DMF with 0.5 % boric acid mixture and reaction was monitored using Kaiser or ninhydrin test. After the reaction complies, the resin is washed with DMF/0.5% formic acid followed by DMF washings were monitored by Chloranil test.
The rest of the amino acids were coupled as per the sequence using following coupling conditions:
Fmoc-AA-OH/Boc-AA-OH, coupling agent and additive were dissolved in DMF and the reaction mixture was added to the resin, stirred for 1.5 – 2.0 h and reaction monitored using Kaiser or ninhydrin test. After reaction complies resin was washed with DMF.
Fmoc-Axx-OH/ Boc-Axx-OH/Fragment, coupling agent, additive details given below:
i) Gly is coupled using DIC/OxymaPure
ii) Trp, Gln, Phe, Ile, Ala, Val, Pro, Leu and SC-1 are coupled using HBTU/DIPEA/HOBt.H2O
iv) Ser, Thr, Lys, Aib, Glu, Tyr, and Asp are coupled using HATU/DIPEA/OxymaPure
All Fmoc-deprotections were performed by using 5- 20 % piperidine in DMF with 0.5 % boric acid. Capping of the unreacted sites after coupling of Ile12, Glu3, Aib2 and Tyr1 was performed using acetic anhydride and DIPEA mixture. After completion of linear synthesis, Alloc protecting group on Lys was removed using Pd(PPh3)4 and phenylsilane and stirred at 25-30°C till completion of reaction. After the reaction by complies, solvent was removed using vacuum and resin was washed with DCM wash followed by DMF in 1 % DIPEA and fresh DMF.
Coupling of SC-1 on Lys20 acid was done using HBTU/ HOBt.H2O/ DIPEA in DMF for 2-3 h and reaction was monitored using Kaiser test. After reaction complies resin was washed with DMF to obtain Boc-Tyr1(tBu)-Aib2-Glu3(OtBu)-Gly4-Thr5(tBu)-Phe6-Thr7(tBu)-Ser8(tBu)-Asp9(OtBu)-Tyr10(tBu)-Ser11(tBu)-Ile12-Aib13-Leu14-Asp15(OtBu)-Lys16(Boc)-Ile17-Ala18-Gln19(Trt)-Lys20(AEEA-AEEA-?-Glu(OtBu)-C20-diacid(OtBu))-Ala21-Phe22-Val23-Gln24(Trt)-Trp25(Boc)-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser32(tBu)-Ser33(tBu)-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39(tBu)--Rink amide resin.
Resin was washed with DMF followed by DCM, MeOH and MTBE and dried. Cleavage cocktail mixture of TFA/TIPS/Phenol/H2O (80: 5: 10: 5 %) was added to the resin and stirred for 3-4 h. After reaction complies, the reaction mixture was filtered, filtrate was evaporated and crude peptide was isolated using chilled MTBE. Crude peptide was washed with MTBE and dried to obtain crude Tirzepatide. Crude: 170 g and product content 42.5 g. The crude peptide was purified in a manner analogous to that described in example-2 to get pure Tirzepatide API. Yield: 18.36 g.

Dated this: 7th Aug. 2023 Dr. S. Ganesan
Alembic Pharmaceutical Ltd.

CLAIMS:We claim:

1. A compound wherein the compound is any one of the amino acid sequences selected from the group consisting of SEQ ID: 1, SEQ ID: 2, SEQ ID: 3, SEQ ID: 6, SEQ ID: 7, SEQ ID: 8, SEQ ID: 9, SEQ ID: 10, SEQ ID: 11 or a pharmaceutically acceptable salt thereof.
2. A process for the preparation of Tirzepatide or a pharmaceutically acceptable salt thereof, comprising use of at least one compound selected from group consisting of SEQ ID: 1, SEQ ID: 2, SEQ ID: 3, SEQ ID: 4, SEQ ID: 5, SEQ ID: 6, SEQ ID: 7, SEQ ID: 8, SEQ ID: 9, SEQ ID: 10, SEQ ID: 11, SEQ ID: 12, SEQ ID: 13, SEQ ID: 14 or a pharmaceutically acceptable salt thereof.
3. A process for the preparation of preparation of Tirzepatide comprising;
a. loading of Fmoc-Ser39-NH2 to resin;
b. sequential coupling of amino acids based on the sequence of peptide backbone of Tirzepatide till Tyr1 in the presence of coupling agent to obtain SEQ ID: 1 wherein Xn is selected from the group consisting of Alloc, Mtt, Dde, Mmt and ivDde;
c. deprotection of protecting group (Xn) from Lys20 to obtain SEQ ID: 2, reacting with side chain protected or un-protected fragment eicosanedioic acid-?-Glu-AEEA-AEEA-OH OR sequentially reacting with AEEA, AEEA, ?-glutamic acid(OtBu), eicosanedioic acid(OtBu) to obtain SEQ ID: 3 and converting the SEQ ID: 3 to Tirzepatide.
4. A process for the preparation of preparation of Tirzepatide comprising;
b. loading of Fmoc protected serine to a resin;
c. sequential coupling of amino acids based on the sequence of peptide backbone of Tirzepatide till Gln19 in the presence of coupling agent
d. coupling of Tyr1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Ile12-Aib13-Leu14-Asp15-Lys16-Ile17-Ala18-OH (SEQ ID: 11) in the presence of coupling agent
e. deprotection of protecting group (Xn) from Lys20 and converting the resulting peptide to Tirzepatide.
5. A process for the preparation of preparation of Tirzepatide comprising;
e. loading of Fmoc protected serine to a resin;
f. sequential coupling of amino acids based on the sequence of peptide backbone of Tirzepatide till Gln19 in the presence of coupling agent
g. coupling of Lys16-Ile17-Ala18-OH (SEQ ID: 12) in the presence of coupling agent
h. sequential coupling of amino acids based on the sequence of peptide backbone of Tirzepatide till Tyr1 in the presence of coupling agent
f. deprotection of protecting group (Xn) from Lys20 and converting the resulting peptide to Tirzepatide.
6. A process for the preparation of preparation of Tirzepatide according to any of claims 1-5, wherein resin is selected from group comprising of comprising Rink amide resin (RAR), Seiber amide resin, Chlorotrityl resin (CTC).
7. A process for the preparation of preparation of Tirzepatide according to any of claims 1-5, wherein coupling agent is selected from group comprising of DIC/HOBt.H2O; DIC/OxymaPure; HBTU/HOBt.H2O/DIPEA, HBTU/OxymaPure/DIPEA; HBTU/Oxy-B/DIPEA; HATU/HOAt/DIPEA or PyBOP/ HOBt.H2O/DIPEA.
8. A process for the preparation of preparation of Tirzepatide according to any of claims 1-5, wherein deprotection of protecting group (Xn) from Lys20 comprises using hydrazine hydrate when Xn group is Dde, using HFIP, TFE, TES when Xn group is Mtt, using Pd(PPh3)4 and phenylsilane when Xn group is Alloc.
9. A process for the preparation of preparation of Tirzepatide according to any of claims 1-5, wherein conversion to Tirzepatide comprises concomitant cleavage from the resin and side chain protecting group removal using mixture of TFA, TIPS, Phenol, H2O.

Dated this: 7th Aug. 2023 Dr. S. Ganesan
Alembic Pharmaceutical Ltd.