DESC:CROSS REFERENCE TO RELATED APPLICATIONS
This is a cognate application of the provisional patent application No. 202241073671 filed on 19 Dec 2022, and provisional patent application No. 202341072758 filed on 26 Oct 2023.

FIELD OF THE INVENTION
The present application relates to processes for the preparation of tirzepatide, its intermediate peptides, and pharmaceutical compositions comprising tirzepatide.

BACKGROUND OF THE INVENTION
Tirzepatide a dual glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) receptor agonist that is developed by Lilly Pharmaceuticals for the treatment of type 2 diabetes. Tirzepatide has positive effects on blood sugar control and weight loss, and the tolerance is improved with dose escalation. Tirzepatide is a 39-amino-acid modified peptide with a C20 fatty diacid moiety that is attached to a lysine, and has the following chemical structure:

Tirzepatide is approved by the US FDA as an adjunct to diet and exercise to improve glycemic control in adults with type 2 diabetes mellitus.
Tirzepatide, its synthetic process and its pharmaceutical compositions are described in US patent No. 9,474,780 (US ‘780). The synthetic approach of tirzepatide described in US ‘780 is a solid phase peptide synthesis involving linear peptide synthesis, and then coupling the side-chain to lysine in a step-wise process.
US Patent No. 11,357,820 describes pharmaceutical compositions of tirzepatide.
PCT Application WO 2020/159949 A1 describes solid phase peptide synthesis and liquid phase peptide synthetic processes for preparation of tirzepatide and intermediate amino-acid fragments.
PCT Application WO 2023/089594 A1 describes processes for preparation of Tirzepatide and intermediate peptide fragments.
Chinese patent applications (CN110903355 A, CN112110981 A and CN112592387 A) also describe synthetic processes and purification methods of tirzepatide.
There remains a need to provide commercially viable and advantageous processes for synthesis of tirzepatide and its pharmaceutical compositions.

SUMMARY OF THE INVENTION
The present application provides processes for preparation of tirzepatide, its intermediate compounds and pharmaceutical compositions containing tirzepatide prepared by the process of the application.
In one aspect the present application provides a process for preparation of Tirzepatide or a pharmaceutically acceptable salt thereof, comprising:
(a) preparing an intermediate peptide having 19 amino acids of Formula XV,
(b) reacting the intermediate peptide having 19 amino acids with ivDde-Lys(Fmoc)-OH to form an intermediate peptide having 20 amino acids of Formula XIV

(c) deprotecting the Fmoc group of the intermediate peptide having 20 amino acids of Formula XIV to form deprotected form of the intermediate peptide of Formula XIV’

(d) reacting the intermediate peptide of Formula XIV’ with 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid to form the intermediate peptide of Formula XIII

(e) deprotecting the intermediate peptide of Formula XIII to form the intermediate peptide of Formula XIII’

(f) reacting the intermediate peptide of Formula XIII’ with 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid to form the intermediate peptide of Formula XII.

(g) deprotecting the intermediate peptide of Formula XII to form the intermediate peptide of Formula XII’

(h) reacting the intermediate peptide of Formula XII’ with Fmoc-Glu-OtBu to form an intermediate peptide of Formula XI

(i) deprotecting the intermediate peptide of Formula XI to form the intermediate peptide of Formula XI’

(j) reacting the intermediate peptide of Formula XI’ with 20-(tert-butoxy)-20-oxoicosanoic acid to form an intermediate peptide of Formula X.

(k) deprotecting the intermediate peptide of Formula X to get the intermediate peptide of Formula X’

(l) coupling remaining 19 amino acid residues in a linear manner using a solid phase peptide synthesizer to form resin bound peptide.

(m) de-blocking the resin bound peptide to form crude tirzepatide, and
(n) purifying the crude tirzepatide.
Wherein,
P is an amine protecting group such as Alloc, ivDde or Mmt;
Side chain is 2-[2-[2-[[2-[2-[2-[[5-tert-butoxy-4-[(20-tert-butoxy-20-oxo-icosanoyl)amino]-5-oxo-entanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetic acid.
Resin is one of 2-CTC resin, Rink Amide resin, Rink Amide AM resin, preferably MBHA Rink Amide resin.
In another aspect the present application provides a process for preparation of Tirzepatide or a pharmaceutically acceptable salt thereof, comprising:
(a) reacting resin bound phenyl alanine with Fmoc-Ala-OH to form resin bound dipeptide, and deprotecting the dipeptide

(b) reacting the dipeptide with Fmoc-Lys(side chain)-OH to form tripeptide,

(c) cleavage of the Fmoc protected tripeptide from the resin

(d) coupling the Fmoc protected trimer to the first resin bound peptide shown below to give an Fmoc protected version of formula X

(e) deprotection of the Fmoc group and coupling remaining amino acid residues in a linear manner using a solid phase peptide synthesizer to form resin bound 39 amino acid peptide.

(d) de-blocking the resin bound peptide to form crude tirzepatide, and
(e) purifying the crude tirzepatide.
Wherein,
Side chain is 2-[2-[2-[[2-[2-[2-[[5-tert-butoxy-4-[(20-tert-butoxy-20-oxo-icosanoyl)amino]-5-oxo-entanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetic acid.
Resin is one of 2-CTC resin, Rink Amide resin, Rink Amide AM resin, preferably 2-CTC resin for the trimer and MBHA Rink Amide resin for tirzepatide.
In one aspect the present application provides an intermediate peptide of Formula V:

In another aspect the present application provides a process for the preparation of tirzepatide, comprising:
a) preparing a dipeptide having lysine and alanine,
b) coupling the dipeptide with the side-chain part to form an intermediate peptide of Formula V,
c) Synthesis of the resin bound peptide sequence shown in Formula XV’ on a suitable amide resin

d) Coupling of the formula V to the intermediate formula XV’ to give the intermediate formula X where P is Fmoc.
e) Deprotection of the Fmoc group on formula X and growing the remaining peptide chain using SPPS.
f) deprotecting the protected amino acids of the peptide and cleavage from the resin to give tirzepatide.

In one aspect the present application provides an intermediate peptide of Formula IV:

In another aspect the present application provides a process for the preparation of an intermediate peptide of Formula IV.
In another aspect the present application provides a process for the preparation of tirzepatide, comprising:
a) preparing a dipeptide having alanine and phenylalanine,
b) coupling the dipeptide with lysine having a side-chain part to form an intermediate peptide of Formula IV,
c) Synthesis of the resin bound peptide sequence shown in Formula XV’’on a suitable amide resin
d) Coupling of the formula IV to the intermediate formula XV’ to give the intermediate formula X where P is Fmoc.

e) Deprotection of the Fmoc group on formula X and growing the remaining peptide chain using SPPS.
f) deprotecting the protected amino acids of the peptide and cleavage from the resin to give tirzepatide.
In one aspect the present application provides an intermediate peptide of Formula III:

wherein P1 is an amino protecting group such as, Fmoc and Alloc or ivDde; and P2 is a carboxyl protecting group such as tert-butyl, and P3 is 2-chlorotrityl or trityl.
In another aspect the present application provides a process for the preparation of an intermediate peptide of Formula III.
In another aspect the present application provides a pharmaceutical composition comprising tirzepatide prepared by the processes described in this application and one or more pharmaceutically acceptable excipient.
These include the building of the side chain directly onto the solid phase during the SPPS process. This is achieved by orthogonal protection of the N-terminal lysine amine using ivDde or Alloc, as well as the use of acid labile orthogonal protecting groups such as Mmt.

DETAILED DESCRITPION OF THE INVENTION
The present application provides processes for the preparation of tirzepatide and its pharmaceutical compositions.
The following abbreviations have been used throughout this document; ACN (acetonitrile), AcOH (acetic acid), Alloc (allyloxycarbonyl), Cbz (benzyl chloroformate), DCC (N,N-dicyclohexylcarbodiimide), CTC (2-chlorotrityl chloride), CTR (2-chlorotrityl resin), DCM (dichloromethane), DCU (1,3-dicyclohexyl urea), Dde (1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl), ivDde (1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)isovaleryl), DEPBT (3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one), DIC (diisopropylcarbodiimide), DIPEA (diisopropylethyamine), DMF (N,N-dimethylformamide), DODT (3,6-dioxa-1,8-octanedithiol), EDC/EDCI (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), EDT (1,2-ethanedithiol), EtOAc (ethyl acetate), Fmoc (fluorenylmethyloxycarbonyl), HOSu (hydroxysuccinimide), HPLC (high performance liquid chromatography), LCMS (liquid chromatography/mass spectroscopy), MBHA (4-methylbenzhydrylamine), MeOH (methanol), Mmt (monomethoxy trityl), MTBE (methyl tert-butyl ether), Mtt (4-methyl trityl), NMR (nuclear magnetic resonance spectroscopy, NMM (N-methylmorpholine), NMP (1-methyl-2-pyrrolidinone), PFP (pentafluorophenol), SPPS (solid phase peptide synthesis), TFA (trifluoroacetic acid), TFE (trifluoroethanol), THF (tetrahydrofuran), TIPS (triisopropylsilane), UPLC (ultra performance liquid chromatography), Boc(tert-butyloxycarbonyl),Fmoc (fluurenylmethyloxycarbonyl), tBu (tert Butyl), Trt (trityl).
Solid phase peptide synthesis (SPPS) of tirzepatide is carried out using a Rink amide MBHA resin swelled in DMF and deprotected using 15% piperidine/ DMF. After the first deprotection the resin is washed with DMF before the first amino acid is coupled. All amino acids are added by Fmoc-SPPS coupling using DIC and ethyl cyanohydroxyiminoacetate (Oxyma) at 3:6:3 (amino acid:DIC:Oxyma) mol equivalence in DMF. The temperatures, times and number of couplings vary depending on the difficulty of the coupling. Subsequent deprotections are carried out in 15% piperidine /DMF. Between coupling and deprotection steps the resin is washed with DMF. These conditions are repeated for each of the deprotection and coupling steps. The number of steps is dependent on how many amino acids are attached to the side-chain when added.
The final crude peptide is cleaved from its resin support and amino acid protecting groups globally deprotected with TFA before UPLC, or LCMS analysis.
In one aspect the present application provides a process for preparation of Tirzepatide or a pharmaceutically acceptable salt thereof, comprising:
(a) preparing an intermediate peptide having 19 amino acids of Formula XV,
(b) reacting the intermediate peptide having 19 amino acids with ivDde-Lys(Fmoc)-OH to form an intermediate peptide having 20 amino acids of Formula XIV

(c) deprotecting the Fmoc group of the intermediate peptide having 20 amino acids of Formula XIV to form deprotected form of the intermediate peptide of Formula XIV’

(d) reacting the intermediate peptide of Formula XIV’ with 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid to form the intermediate peptide of Formula XIII

(e) deprotecting the intermediate peptide of Formula XIII to form the intermediate peptide of Formula XIII’

(f) reacting the intermediate peptide of Formula XIII’ with 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid to form the intermediate peptide of Formula XII.

(g) deprotecting the intermediate peptide of Formula XII to form the intermediate peptide of Formula XII’

(h) reacting the intermediate peptide of Formula XII’ with Fmoc-Glu-OtBu to form an intermediate peptide of Formula XI

(i) deprotecting the intermediate peptide of Formula XI to form the intermediate peptide of Formula XI’

(j) reacting the intermediate peptide of Formula XI’ with 20-(tert-butoxy)-20-oxoicosanoic acid to form an intermediate peptide of Formula X.

(k) deprotecting the intermediate peptide of Formula X to get the intermediate peptide of Formula X’

(l) coupling remaining 19 amino acid residues in a linear manner using a solid phase peptide synthesizer to form resin bound peptide.

(m) de-blocking the resin bound peptide to form crude tirzepatide, and
(n) purifying the crude tirzepatide.
wherein,
P is an amine protecting group such as Alloc, ivDde and Mmt;
Side chain is 2-[2-[2-[[2-[2-[2-[[5-tert-butoxy-4-[(20-tert-butoxy-20-oxo-icosanoyl)amino]-5-oxo-entanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetic acid.
Resin is one of 2-CTC resin, Rink Amide resin, Rink Amide AM resin, preferably MBHA Rink Amide resin.

In another aspect the present application provides a process for preparation of Tirzepatide or a pharmaceutically acceptable salt thereof, comprising:
(a) reacting resin bound phenyl alanine with Fmoc-Ala-OH to form resin bound dipeptide, and deprotecting the dipeptide

(b) reacting the dipeptide with Fmoc-Lys(side chain)-OH to form tripeptide,

(c) cleavage of the Fmoc protected tripeptide from the resin

(d) Purification of Fmoc-Lys(side chain)-Ala-Phe-OH by RP-HPLC.
(e) Synthesis of the resin bound peptide sequence shown in Formula XV’’on a suitable amide resin
(f) Coupling of the formula IV to the intermediate formula XV’ to give the intermediate formula X where P is Fmoc.

(f) Deprotection of the Fmoc group on formula X to give formula X’ and growing the remaining peptide chain using Linear SPPS.

(g) de-blocking the resin bound peptide to form crude tirzepatide, and
(h) purifying the crude tirzepatide.
wherein,
Side chain is 2-[2-[2-[[2-[2-[2-[[5-tert-butoxy-4-[(20-tert-butoxy-20-oxo-icosanoyl)amino]-5-oxo-entanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetic acid.
Resin is one of 2-CTC resin, Rink Amide resin, Rink Amide AM resin, preferably 2-CTC resin for the trimer and MBHA Rink Amide resin for tirzepatide.

In another aspect the present application provides process for preparation of Tirzepatide or a pharmaceutically acceptable salt thereof, comprising use of at least one polypeptide or a pharmaceutically acceptable salt thereof selected from:

P1 is H or Fmoc; P2 is selected from H, ivDde, Alloc or Mmt.

P1 is H or Fmoc; P2 is selected from H, ivDde, Alloc and Mmt.

P1 is H or Fmoc; P2 is selected from H, ivDde, Alloc and Mmt.

P2 is selected from H, ivDde, Alloc and Mmt.

In one aspect the present application provides an intermediate peptide of Formula V:

wherein P1 is an amino protecting group such as Fmoc or Ivdde and P2 is a carboxyl protecting group such as tert-butyl, and 2-chlorotrityl.
In another aspect the present application provides a process for the preparation of an intermediate peptide of Formula V, comprising:
a) reacting an ester of alanine or its salt with a compound of Formula VI in the presence of a coupling reagent and a suitable base to form an ester of compound of Formula V named Formula VII.

wherein P1 is an amino protecting group such as Fmoc, ivDde, Mmt and Alloc; P2 is a carboxyl protecting group such as t-butyl, and 2-chlorotrityl; and R is a substituted or an unsubstituted C1-4 alkyl; and
b) saponification of the ester using calcium iodide and a suitable base to form the compound of Formula V.

Step-1 of the process involves the coupling of an alanine ester or a salt thereof with a compound of Formula VI in the presence of a coupling reagent and a suitable base to form an ester of compound of Formula V.
The alanine ester is selected from the group comprising of methyl ester, ethyl ester, tert-butyl ester and benzyl ester. In one aspect the alanine ester is methyl ester.
The base is selected from the group comprising of sodium carbonate, potassium carbonate, triethylamine and DIPEA. The coupling reagent is selected from the group comprising DCC, DIC, EDC and DEPBT.
The alanine methyl ester, the base and the coupling reagent are added to a suitable solvent such as THF, DMF, ACN, NMP and DCM. The reaction mixture may be stirred for about 30 minutes to about 10 hours. After completion of the reaction the mixture is filtered and the solution containing the product is concentrated. The residue may be purified by column chromatography.
Step-2 involves the reaction of the Step-1 product with calcium iodide and a suitable base in a suitable solvent. The base used is selected from the group comprising LiOH, NaOH and KOH. The solvent used is selected from the group comprising of acetone, THF, acetonitrile and water, or a mixture thereof.
The Step-1 product, base and calcium iodide are added to a suitable solvent and stirred for about 30 minutes to about 10 hours. After completion of the reaction the reaction mixture may be concentrated and acidified with dil. aqueous HCl and the mixture may be extracted with DCM and the DCM layer may be concentrated. The crude acid may be purified by column chromatography.

In another aspect the present application provides a process for the preparation of an intermediate peptide of Formula V, comprising:
a) reacting alanine or its salt with a lysine or a salt thereof in presence of a coupling reagent and a suitable base to form a dipeptide of Formula VIII

b) optionally deprotecting the dipeptide of Formula VIII,
c) coupling the dipeptide of Formula VIII with tirzepatide side chain to form the intermediate peptide of Formula V.

wherein P1 is an amino protecting group such as Boc; P2 is a carboxyl protecting group such as tert-butyl, or 2-chlorotrityl, and P3 is Fmoc, ivDde or Alloc.

Step-1 of the process involves reaction of lysine with alanine or a salt thereof in the presence of a suitable coupling agent such as DEPBT and a suitable base as DIPEA and a suitable solvent such as THF. The amino acid lysine may be taken as Fmoc protected lysine and the alanine may be taken as tert.-butyl alanine hydrochloride.
Fmoc-lysine, THF, DEPBT and DIPEA are mixed and stirred then alanine tert.-butyl ester hydrochloride is added and stirred until Fmoc-lysine is completely reacted. The reaction mixture may be concentrated, and the residue is purified to give the dipeptide.
Step-2 involves deprotection of the dipeptide. Boc and t-butyl groups are deprotected using suitable acid such as dilute HCl. The dipeptide obtained in Step-1 is dissolved in a suitable solvent such as DCM and the mixture is treated with 4 M HCl in dioxane and stirred for about 30 minutes. The product may be isolated by filtration.
Step-3 involves coupling the dipeptide of Formula VIII with the tirzepatide side chain to from the intermediate peptide of Formula V.

In one aspect the present application provides before reacting, the side chain may be activated by reacting it with a suitable activating agent such PFP or HOSu. The activated side chain then reacted with the dipeptide in presence of a suitable base such as N-methyl morpholine and a suitable solvent such as THF to form the intermediate peptide of Formula V.
The activated side chain, THF, the dipeptide and the base are mixed and stirred for about 3 hours. The reaction mixture may be concentrated and purified to get the intermediate peptide of Formula V.

In another aspect the present application provides a process for the preparation of an intermediate peptide of Formula V using a fragment based approach, comprising:

a) reacting alanine or its salt with a lysine or a salt thereof in presence of a coupling reagent and a suitable base to form a dipeptide of Formula VIII
b) optionally deprotecting the dipeptide of Formula VIII,
c) reacting a dipeptide of Formula VIII with a compound of Formula XII to form a compound of Formula XI, where formula XII is preactivated with either PFP or HOSu.

d) deprotection of the compound of formula XI using acidic conditions
e) reacting the compound of deprotected Formula XI with a compound of Formula X to form a compound of Formula V, where formula X is preactivated with either PFP or HOSu.

wherein P1 is the amino protecting group Fmoc; P2 is a carboxyl protecting group such as t Butyl and 2-chlorotrityl

The amino dipeptide may be taken as Fmoc protected dipeptide and the compound of formula XII may be taken as an activated form of compound of formula XII-A or compound of formula XII-B as shown in below:

The dipeptide, THF and NMM are mixed and stirred, then the compound of Formula XII in its activated form is added and stirred until the dipeptide is completely reacted. The reaction mixture may be concentrated, and the residue is purified to get the compound of Formula XI in the Boc protected form.

The compound of Formula XI may be deprotected. Boc groups are deprotected using suitable acid such as dilute HCl. The compound of Formula XI obtained in Step-1 is dissolved in a suitable solvent such as DCM and the mixture is treated with 4 M HCl in dioxane and stirred for about 30 minutes. The product may be isolated by filtration to get the compound of Formula XI-A

Step-2 involves coupling the compound of Formula XI or XI-A with compound of Formula X to form the intermediate peptide of Formula V.
In one aspect the present application provides before reacting, the compound of Formula X may be activated by reacting it with a suitable activating agent such as PFP or HOSu. The activated compound of Formula X is then reacted with the compound of Formula XI or XI-A in the presence of a suitable base such as N-methyl morpholine and a suitable solvent such as THF to form the intermediate peptide of Formula V.
The activated compound of Formula X, THF, the compound of Formula XI or XI-A and the base are mixed and stirred for about 3 hours. The reaction mixture may be concentrated and purified to get the intermediate peptide of Formula V.

In another aspect the present application provides a solid phase peptide synthesis of an intermediate dipeptide of Formula V, comprising:
a) reacting 2-CTR with Fmoc-Ala-OH to form Fmoc-Ala-CTR and deprotecting it using piperidine to form NH2-Ala-CTR,
b) reacting NH2-Ala-CTR with Fmoc-Lys(SC)-OH to form protected dipeptide,
c) Cleaving the protected dipeptide from the resin to form the dipeptide of Formula V

In another aspect the present application provides a solid phase peptide synthesis of an intermediate tripeptide of Formula IV, comprising:
a) reacting 2-CTR with Fmoc-Phe-OH to form Fmoc-Phe-CTR and deprotecting it using piperidine to form NH2-Phe-CTR,
b) reacting 2-CTR phenylalanine with Fmoc-Ala-OH to form Fmoc-Ala-Phe-CTR and deprotecting it using piperidine to form NH2-Ala-Phe-CTR,
c) reacting NH2-Ala-Phe-CTR with Fmoc-Lys(SC)-OH to form a tripeptide,
d) Cleaving the tripeptide from the resin to form the tripeptide of Formula IV

In another aspect the present application provides a process for the preparation of side-chain coupled dipeptide. The process is schematically shown below:

In another aspect the present application provides a solid phase peptide synthesis of an intermediate tetrapeptide of Formula III, comprising:
a) reacting 2-CTR with Fmoc-Val-OH to form Fmoc-Val-CTR and deprotecting it using piperidine to form NH2-Val-CTR,
b) reacting 2-CTR Valine with Fmoc-Phe-OH to form Fmoc-Phe-Val-CTR and deprotecting it using piperidine to form NH2-Phe-Val-CTR,
c) reacting NH2-Phe-Val-CTR with Fmoc-Ala-OH to form Fmoc-Ala-Phe-Val-CTR and deprotecting it using piperidine to form NH2-Ala-Phe-Val-CTR,
d) reacting NH2-Ala-Phe-CTR with Fmoc-Lys(SC)-OH to form a tetrapeptide,
e) Cleaving the tetrapeptide from the resin to form the tetrapeptide of Formula III:

In another aspect the present application provides a process for the preparation of
tirzepatide, comprising
a) preparing the intermediate dipeptide of Formula V, or tripeptide of Formula IV by the process described in this application and
b) growing the entire peptide by SPPS on a suitable amide resin using the intermediate peptide Formula V or Formula IV, and
c) deprotecting the protected amino acids of the peptide and cleaving from the resin.

In another aspect the present application provides a process for the preparation of
tirzepatide, comprising:
a) preparing an intermediate peptide of Formula VIII having 20 amino acids including lysine where the epsilon amine has a suitable amino protecting group such as Dde, ivDde, Mmt, Mtt or Alloc, which is orthogonal to the Fmoc group protecting the N-terminal amine of the lysine amino acid.
b) coupling the side chain to lysine of the intermediate peptide to form the intermediate peptide of Fmoc-Lys(side chain)AFVQWLIAGGPSSGAPPPS-Rink Amide Resin,
c) growing the entire peptide on the intermediate peptide, and
d) deprotecting the protected amino acids of the peptide.

The process is schematically described below:

In another aspect the present application provides a process for the preparation of tirzepatide, comprising:
a) preparing an intermediate peptide of Formula IX having 19 amino acids,
b) coupling of the side-chain containing lysine with the intermediate peptide of Formula IX to form the intermediate peptide of Formula VII,
c) growing the entire peptide on the intermediate peptide of Formula VII, and
d) deprotecting the protected amino acids of the peptide.
The process is schematically described below:

In another aspect the present application provides a process for the preparation of tirzepatide. The process is schematically described below:

In another aspect the present application provides a process for the preparation of Tirzepatide. The process is schematically described below:

In another aspect the present application provides a process for the preparation of Tirzepatide. The process is schematically described below

a) preparing an intermediate peptide on a suitable amide resin having 39 amino acids including lysine where the epsilon amine has a suitable amino protecting group such as Dde, ivDde, Mmt, Mtt or Alloc, which is orthogonal to the Fmoc group protecting the N-terminal amines of the amino acids used in the synthesis and to the side chain protecting groups of the amino acids used in the synthesis and where the N terminus of the peptide is Boc protected.
b) Selective deprotection of the epsilon amine of the lysine and addition of the side chain though normal SPPS conditions.
c) deprotection of amino acid side chains and cleavage from the resin.

The process is schematically described below:

In another aspect the present application provides pharmaceutical compositions comprising tirzepatide prepared by the processes described in this application and one or more pharmaceutically acceptable excipient.

DEFINITIONS
The following definitions are used in connection with the present application unless the context indicates otherwise.
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%, more preferably within a range of ±2%, still more preferably within a range of ±1 % of its value. For example, "about 10" should be construed as meaning within the range of 9 to 11, preferably within the range of 9.5 to 10.5, more preferably within the range of 9.8 to 10.2, and still more preferably within the range of 9.9 to 10.1.
All percentages and ratios used herein are by weight of the total composition and all measurements made are at about 25°C and about atmospheric pressure, unless otherwise designated. All temperatures are in degrees Celsius unless specified otherwise. As used herein, “comprising” means the elements recited, or their equivalents in structure or function, plus any other element or elements which are not recited. The terms “having” and “including” are also to be construed as open ended. All ranges recited herein include the endpoints, including those that recite a range “between” two values. Whether so indicated or not, all values recited herein are approximate as defined by the circumstances, including the degree of expected experimental error, technique error, and instrument error for a given technique used to measure a value.
Certain specific aspects and embodiments of the present application will be explained in greater detail with reference to the following examples, which are provided only for purposes of illustration and should not be construed as limiting the scope of the application in any manner. Reasonable variations of the described procedures are intended to be within the scope of the present invention. While particular aspects of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

EXAMPLES
Example-1: Preparation of Fmoc-Lys(SC)-Ala-OMe
Fmoc-Lys(SC)-OH (9.6 g, 7.8 mmol) was dissolved in THF (50 mL). DEPBT (5.3 g, 17.7 mmol) and DIPEA (4.5 mL, 25.8 mmol) were added. Followed by alanine methylester hydrochloride (1.3 g, 9.3 mmol) and the reaction was stirred at RT overnight. The reaction had gone to approx. 94% conv. in 18 hours. The reaction was left to run for an additional 4 hours. The reaction mixture was concentrated under reduced pressure to remove most of the THF. The residue was dissolved in DCM (Ca 100 mL). The DCM solution was washed with 1 M HCl (2 x100 mL). The combined acidic washes were back-extracted with DCM (30 mL). DCM solutions were concentrated under reduced pressure to remove most of the DCM. 14.5 g of crude residue obtained.

Example-2: Saponification of Fmoc-Lys(SC)-Ala-OMe to form Fmoc-Lys(SC)-Ala-OH
Crude Fmoc-Lys(SC)-Ala-OH, (Ca 11 g, max 6.9 mmol) was dissolved in acetone (125 mL), and water (50mL). To this solution was added CaI2 (23 g, 23.8 mmol, 3.45eq.) and 2 M NaOH (5.2 mL, 10.35 mmol, 1.5 eq.). The reaction was stirred at RT for 2 hours, but no conversion was observed. Additional CaI2 (25 g) was added to the reaction (total now 48 g, 163 mmol, ~23 eq.). Additional 2 M NaOH (3 mL) was slowly added over 48 hours. Analysis now showed 84% of related material is product. The reaction mass was acidified with 2 M HCl, to pH 5. Most of the acetone was removed under reduced pressure. The concentrated mixture was further acidified to pH 3 with addition of 2 M HCl, and this acidic mixture was extracted with MTBE (150 mL). The aqueous layer was then extracted with DCM (150 mL). The DCM layer was concentrated under reduced pressure to give a thick yellow oil; some solvent was still present (5 g).
Example-3: Solid Phase Peptide Synthesis of Fmoc-Lys(SC)-Ala-OH

Loading of 2-CTC Resin:
Fmoc-Ala-OH.H2O (1.2 equiv. to the resin, 19.2 mmol, 6.30 g) and DIPEA (6 equiv. to amino acid, 76.8 mmol, 20 mL) were dissolved in dry DCM (100 mL) in a 250 mL two-neck flask under nitrogen. Once all the Fmoc-Ala-OH.H2O was dissolved, the solution was transferred via cannula to a separate 500 mL two-neck flask, containing 2-CTC resin (16 mmol, 10 g). The suspension was stirred for 3 hours. At the end of this time, the resin was washed with DCM/MeOH/DIPEA (17:2:1, 3 x 100 mL), 3 x DCM (50 mL), DMF (2 x 50 mL), DCM (2 x 100 mL) and dried under a flow of N2 for 1 hour in a manual SPPS vessel. The resin was treated with piperidine 15% in DMF (100 mL) for an hour, washed 3 x DMF (100 mL), 3 x DCM (100 mL) and dried overnight under a flow of N2.
Kaiser testing was conducted on the resin and gave an intense blue colour (positive). Loading with alanine, successful (10.80 g).

Coupling of Fmoc-Lys(SC)-OH:
A peptide synthesiser was charged with the required amounts of 0.20 M Fmoc-Lys(SC)-OH, 1.0 M DIC and 0.5 M Oxyma/DMF solution. 2-CTC Resin, previously manually loaded with alanine (5.6 g, 0.90 mmol/g, assumed) was charged and SPPS performed. The resin bound peptide was off-loaded, washed with DCM (150 mL) and dried overnight under N2 flow.

Isolation of the Peptide:
Resin bound peptide (100 mg) was weighed into a HPLC vial. TIPS (30 µL), DODT (30 µL) and water (20 µL) were added. After 2 min, TFA (900 µL) was added. The mixture was shaken (thermoshaker) at RT for 60 min. The reaction mixture was filtered using a syringe filter (20 µm membrane), and the filtrate added to cooled MTBE (30 mL) held in a centrifuge tube. The tube was sealed, and the mixture cooled in an ice bath for 20 min. The contents were split into six 5 mL Eppendorf tubes. The samples were centrifuged (8000 x g) for 10 min. The liquors were decanted, and a viscous oil was obtained. A few mg of the oil was dissolved in ACN: water 1:1 and analysed by LCMS. LCMS showed the desired dimer.

Cleavage with TFE/DCM:
Resin bound peptide (10.1 g) was treated with TFE/DCM (2:8, 100 mL) at RT for 3 x 1 hour in a manual SPPS peptide vessel (stirred through nitrogen bubbling). The resin bound peptide was removed by filtration and washed with more cleavage mixture (3 x 100 mL). The solution was evaporated to dryness and precipitation of protected dimer was attempted in MTBE and then subsequently diethyl ether, but no precipitation was observed. The solution was concentrated under reduced pressure to give a viscous oil. The oil was redissolved in DCM and evaporated to give a 'sticky' off white solid (3.90 g, 40% yield).

Example-4: Solid Phase Peptide Synthesis of Fmoc-Lys(SC)-Ala-Phe-OH

DMF solutions of 0.44 M Fmoc-Ala-OH (20 mL), 1.0 M DIC (17.5 mL) and 0.5 M Oxyma (17.5 mL) were stirred in a 100 mL RBF for 25 min and added to the reaction vessel just prior to commencement of SPPS. A peptide synthesiser was charged with the required amount of 0.20 M Fmoc-Lys(SC)-OH/DMF solution. 2-CTC Resin previously manually loaded with phenylalanine (2.73 g, 1.1 mmol/g assumed) was charged and SPPS performed. The resin bound peptide was off-loaded, washed with 100 mL of DCM and dried under a flow of N2 overnight.

Resin bound tripeptide (100 mg) was weighed into a HPLC vial. TIPS (30 µL), DODT (30 µL) and water (20 µL) were added. After 2 min, TFA (900 µL) was added. The mixture was shaken at RT for 1 hour. The reaction mixture was filtered using a syringe filter (20 ?m membrane), and the filtrate added to cooled MTBE (30 mL) in a centrifuge tube. The tube was sealed, and the mixture cooled in an ice bath for 20 min. The contents were split into six 5 mL Eppendorf tubes. The samples were centrifuged (8000 x g) for 10 min. The liquors were decanted, concentrated under reduced pressure and a viscous oil obtained. A few mg of the oil was dissolved in ACN/water 1:1 and analysed by LCMS. LCMS showed the desired dimer.

Example-5: Preparation of Tirzepatide using Fmoc-Lys(SC)-Ala-Phe-OH (Compound of Formula IV)
A peptide synthesiser was charged with required amounts of 0.44 M amino acids, Fmoc-Lys(SC)-Ala-Phe-OH, 1.0 M DIC, 0.5 M Oxyma, 15% piperidine/DMF solution and SPPS performed. Once the run was complete, the resin bound peptide was off-loaded, washed with DCM (100 mL) and dried under a flow of N2 overnight.

Resin bound peptide (120 mg) was weighed into a HPLC vial. Phenol (40 mg), EDT (40 µL), TIPS (40 µL) were added. TFA (800 µL) was added. The mixture was shaken at 40oC for 1 hour. The mixture was washed with MTBE, and a sample analysed by LCMS. 22.79% mass match by for tirzepatide by Mass Spec.
Example-6: Preparation of Fmoc-Lys-Ala-OtBu

Fmoc-Lys(Boc)-OH (1.02 g, 2.18 mmol) was dissolved in THF (6 mL). DEPBT (1.47 g, 4.92 mmol) and DIPEA (1.4 mL, 8.13 mmol). The reaction was stirred for 5 min. Alanine tert-butyl ester hydrochloride (0.48 g, 2.61 mmol) was added and the reaction stirred at RT overnight. A sample was analysed by LC/MS, (no starting material observed). The reaction mixture was concentrated under reduced pressure to remove most of the THF. The residue was dissolved in EtOAc (15 mL). The EtOAc solution was washed with 1M HCl (10 mL), brine (10 mL), sat. Na2CO3 (10 mL), & brine (10 mL). The EtOAc solution was concentrated under reduced pressure to give a yellow gum (1.89 g, crude). A sample was analysed by LC/MS and the titled product was observed at 8.79 min (71% purity).

Example-7: Boc and tert-Butyl deprotection of Fmoc-Lys-Ala-OtBu

tert-Butyl-N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(tert-butoxycarbonyl)-L-lysyl-L-alaninate (7.15 g, 12 mmol) was dissolved in dichloromethane (50 mL). 4M HCl in dioxane (30 mL, 120 mmol) was added and stirred overnight. An in process check by LC/MS indicated no starting material. The reaction mixture was concentrated under reduced pressure to give a residue. To the residue was added MTBE (10 mL) and sonicated for 10 min. The suspension was filtered under vacuum and the solid washed with heptane. The solid was further dried under vacuum to yield a product as a white solid (5.20g, 90% yield). A sample was analysed by LC/MS and the titled product was observed at 6.14 min, (97% purity).

Example-8: Activation of t-Bu-C20-Glu(PEG4)-tBu with PFP

To a stirred solution of side chain (2.54 g, 2.90 mmol), pentafluorophenol (0.61 g, 3.34 mmol) in DCM (50 mL) was added DCC (0.75 g, 3.64 mmol) followed by NMM (1 mL, 9.1 mmol). The mixture was stirred for 3 hours. The mixture was filtered using a sintered glass funnel under vacuum. The filtrate was diluted with DCM (100 mL) and stirred with 11% w/w citric acid solution (50 mL) for 5 min. The organic phase was partitioned, dried over MgSO4 and concentrated under reduced pressure to afford a red gum. The crude product was purified by column chromatography. Isolated fractions were concentrated under reduced pressure to yield product (2.35 g, 74% yield) as a red/purple gum. A sample was analysed by LC/MS and the titled product observed at 10.39 min, (48% purity).

Example-9: Coupling of side chain PFP and Fmoc-Lys-Ala-OH

Fmoc-Lys-Ala-OH (3.17 g, 6.66 mmol) was ground with a spatula and added to a stirred solution of side chain (5.79 g, 5.57 mmol) in THF (200 mL) at 5 °C, forming a white suspension. NMM (1.22 mL, 11.1 mmol) was added, and the mixture stirred at 5°C overnight. Acetic acid (2.00 mL) was added, the solvents removed under reduced pressure and purified by column chromatography. The fractions were analysed by LC/MS. Fractions with product were combined and concentrated under reduced to afford a light-yellow gum (8.00 g). Over 100% yield obtained, as product contained residual acetic acid. A sample was analysed by LC/MS, and titled product was observed at 9.87 min, (94% purity).

Example-10: Activation of Boc-AEEA-AEEA-OH to OSu Ester

EDCl (3.40 g, 21.9 mmol) was added to a stirred solution of 2,2-dimethyl-4,13-dioxo-3,8,11,17,20-pentaoxa-5,14-diazadocosan-22-oic acid (5.96 g, crude) in DCM (75 mL). HOSu (2.0 g, 17.5 mmol) Was added, and the mixture stirred overnight. To the mixture was added water (100 mL) and then charged to a separating funnel. The organic layer was separated, dried by MgSO4 and concentrated under reduced pressure to yield crude product. The crude was purified by column chromatography. Fractions with product were concentrated under reduced pressure to afford a colourless oil (2.9 g, crude). The product contained residual acetic acid (23.8% w/w, 1H NMR). A sample was analysed by LC/MS and titled product was observed at 6.47 min (17% purity). Note: Product hydrolysed on the analytical column during LCMS analysis and the hydrolysed product observed at 6.06 min (7% purity).

Example-11: Coupling of Boc-AEEA-AEEA-OSu and Fmoc-Lys-Ala-OH

Fmoc-Lys-Ala-OH (1.40 g, 3.00 mmol) was added to a stirred solution of side chain OSu (2.02 g, crude) in THF (50 mL) at 5 °C forming a white suspension. NMM (0.50 ml, 9.10 mmol) was added, and the mixture stirred at 5 °C overnight. Volatiles were then evaporated, water (80 mL) added, and the pH adjusted to 11.0 with 2 M NaOH. DCM (80 mL) was then added, and the mixture transferred to a separating funnel. The aqueous phase was separated and adjusted to pH 2 with 4 M HCl, and then extracted with DCM (3 x 75 mL). The combined organic phases were dried by MgSO4 and concentrated under reduced pressure to afford (2.64 g, <100% yield) as a colourless gum. A product sample was analysed by LC/MS and titled product was observed at 7.29 min (56% purity).

Example-12: Activation of tBuO-C20-OH with HOSu

OtBu-C20-OH (15.03 g, 37.7 mmol) and HOSu (6.58 g, 56.6 mmol) were dissolved in THF (100 mL). DCC (7.78 g, 37.7 mmol) was added, and the reaction stirred at RT for 2 hours. The precipitation observed was most likely DCU. The solid was removed by filtration with THF washing. The THF was then removed under reduced pressure, the residue dissolved in EtOAc (100 mL) and transferred to a separating funnel. The solution was washed with 0.5 M HCl (100 mL), water (100 ml) and brine (50 ml). The organic phase was dried (MgSO4), filtered and concentrated under reduced pressure. The residue was dissolved in EtOH (200 mL) with gentle warming. Undissolved material was filtered off and dried under vacuum. A sample was submitted for NMR and LC/MS analysis. The filtrate was left to recrystallize at room temp with gentle stirring overnight. The precipitate was filtered and dried under vacuum, and then on a rotary evaporator. Both products were analytically similar and therefore were combined to yield the titled product (13.9 g, 74% yield). A sample was analysed by 1H NMR.

Example-13: Coupling of OtBu-C20-OH with Glu-OtBu

C20-OSu (13.9 g, 28.1 mmol) in THF (100 mL) was added to a stirred solution of Glu-OtBu (7.32 g, 36 mmol) in water (100 ml) over the course of 20 minutes. The mixture was stirred for a further hour. An in-process check indicated incomplete reaction. Additional Glu-OtBu (7.17 g) in water (100 mL) was added and the reaction mixture stirred for an additional 3 hours. Volatiles were removed under reduced pressure and product crashed out. To the mixture was added EtOAc (100 mL) and transferred to a separating funnel. The organic layer was separated. The aqueous phase was washed with EtOAc (3 x 70 mL) and the combined organic layers were washed with brine (100 mL), dried (MgSO4) and concentrated under reduced pressure to afford crude product (14.0 g) as a white suspension (solid was not dry). A sample was analysed by LC/MS, and the target was observed at 10.21 min (23% purity).

Example-14: Activation of OtBu-C20-Glu-OH with HOSu

OtBu-C20-OH (14.0 g, crude) and HOSu (8.29 g) were stirred in ACN (200 mL). EDC (11.18 g) was then added in a single portion and the mixture stirred at RT overnight. Water (100 mL) was then added and stirred for 5 hours. Initially a hazy solution formed, followed by slow crystallisation of the product. The solid was isolated by filtration and then purified by column chromatography. Fractions with product were concentrated under reduced pressure and product isolated as a white solid (11.2 g). The product was recrystallized by dissolving in minimum amount of ethanol with gentle warming. The dissolved material was cooled in an ice-bath. A precipitate was formed, filtered and dried under reduced pressure to yield product (5.71 g, 35% yield) as a white solid. A sample was analysed by LC/MS and the product was observed at 10.36 min (93% purity).

Example-15: Boc deprotection of Lys-(AEEA-AEEA-Boc)-Ala

Lys-(AEEA-AEEA-Boc)-Ala (2.52 g, crude) was dissolved in DCM (30 mL). 4 M HCl in dioxane (6 mL) was added and stirred for 2 hours. A thick gum began forming in the bottom of the flask and was difficult to stir. The mixture was evaporated to dryness under reduced pressure, to give a crude colourless gum (2.42 g). Some of the gum foamed and solidified into a pale yellow solid. Slightly over 100% yield was obtained. A sample was analysed by LC/MS and the target was observed at 5.40 min.

Example-16: Coupling of Fmoc-Lys-(AEEA-AEEA-NH2)-Ala-OH and tBu-C20-Glu(OSu)-OtBu

tBu-C20-Glu(OSu)-OtBu (2.11 g, 3.1 mmol) was added to a stirred solution of Fmoc-Lys-(AEEA-AEEA-NH2)-Ala (2.26 g, 3.1 mmol) in THF (100 mL) and water (30 mL) at 5 °C, forming a clear light-yellow solution. NMM (2 mL, 18.19 mmol) was added, and the mixture stirred at 5 °C overnight. After the solvents were removed under reduced pressure, the product was dissolved in DCM and purified by column chromatography. Fractions with product were combined and concentrated under reduced pressure to afford a light-yellow gum (2.5 g, 44% yield). A sample was analysed by LC/MS and the target was observed at 10.84 min (90% purity).

Example –17: Prep-HPLC purification of Fmoc-Lys(SC)-Ala-OH
Crude Fmoc-Lys(SC)-Ala-OH (2.5 g) was dissolved in 60 mL of 40:60 ACN:Water containing 0.1% NH4OH. This solution was then filtered through 0.22 µm filter. This was loaded onto a pre-equilibrated Gemini C18 10 µm 50x250 mm column. The material was eluted from the column using a gradient of 40->75% B over 40 min with a flow rate of 118 ml/min. MPA was 0.1% NH4OH in water and MPB was 0,1% NH4OH in ACN. The desired product eluted between 60-65% MPB. ACN was removed from the selected fractions under reduced pressure and the resulting aqueous solution was lyophilised. Material (1.6 g) was recovered with a purity of 97.4% by UPLC. UPLC analysis was perfomed using an Aquity CSH phenyl hexyl column (2.1x150 mm). MPA was 10 mM ammonium bicarbonate pH 8.4 and MPB was ACN. The gradient was 55-80% MPB over 23 min. Flow rate was 0.35 mL/min, column temperature was 50°C and detection was UV at 220 nm.

Example-18: Preparation of tirzepatide using Fmoc-Lys(Mmt)-OH
A peptide synthesiser was charged with the required amount of 0.44 M amino acid (including Fmoc-Lys(Mmt)-OH), 1.0 M DIC solution, 0.5 M Oxyma and 15 % piperidine/DMF solution and SPPS performed. After completion, the resin bound peptide was off-loaded, washed with DCM (100 mL) and dried under a flow of N2 overnight.

Mmt group removal
Resin bound peptide was treated with a solution of AcOH/TFE/DCM :1/2/7 (50 vol). The mixture was stirred at RT for 1 hour. The resin bound peptide was filtered, washed with DCM and dried.

Addition of the side chain
A peptide synthesiser was charged with a 0.20 M side chain solution in DMF. Resin bound peptide (after Mmt removal) was charged to the reaction vessel and SPPS performed. After completion, the resin bound peptide was off-loaded, washed with DCM and dried under a flow of N2 overnight.

Resin bound peptide (120 mg) was weighed into a HPLC vial. Phenol (40 mg), EDT (40 µL), TIPS (40 µL) were added. TFA (800 µL) was added. The mixture was shaken at 40 oC for 1 hour. The mixture was washed with MTBE and a sample analysed by LCMS. 70.64% crude mass match for tirzepatide by Mass Spec.

Example-19: Synthesis of Fmoc-Lys(SC)-Ala-Phe-Val-OH

In a peptide synthesiser, 2-CTC resin, previously manually loaded with valine (3.49 g, 0.86 mmol/g assumed, 3.0 mmol) was charged and swelled in DMF. In a 100 mL RBF, DMF solutions of 0.44 M Fmoc-Phe-OH (20 mL), 1.0 M DIC (17.5 mL) and 0.5 M Oxyma (17.5 mL) were stirred for 25 min and then added to the reaction vessel just prior to SPPS. The peptide synthesiser was then charged with the required amounts of 0.44 M Fmoc-Ala-OH and 0.20 M Fmoc-Lys(SC)-OH/DMF solution, and SPPS performed. Once complete, the resin bound peptide was off-loaded, washed with DCM (100 mL) and dried under a flow of N2 overnight.

Resin bound tetrapeptide (100 mg) was weighed into a HPLC vial. TIPS (30 µL), DODT (30 µL) and water (20 µL) were added. After 2 min TFA (900 µL) was added. The mixture was shaken at RT for an hour. The reaction mixture was filtered using a syringe filter (20 ?m membrane), and the liquors added to cooled MTBE (30 mL) in a centrifuge tube. The tube was sealed, and the mixture cooled in an ice bath for 20 min. The contents were split into six 5 mL -Eppendorf tubes. The samples were centrifuged (8000 x g) for 10 min. The liquors were decanted, and a viscous oil was obtained. A few mg of the oil were dissolved in ACN/water 1:1 and analysed by LC/MS. The LC/MS showed the desired product.

Example-20: Preparation of Tirzepatide using ivDde-Lys(Fmoc)-OH

A peptide synthesiser was charged with the required amounts of 0.44 M amino acid (including ivDde-Lys-Fmoc-OH) and side chain components, (AEEA, Glu), 0.20 M C20-diacid, 1.0 M DIC solution, 0.5 M Oxyma and 15% piperidine/DMF solution (all in DMF). After completion, the resin bound peptide was off-loaded, washed with DCM and dried under a flow of N2 overnight. A small aliquot of the corresponding resin bound peptide was isolated and analyzed by LC/MS.

ivDde Group Removal
Resin bound peptide was placed in a manual SPPS vessel and treated with 4% hydrazine monohydrate in DMF (24 vol). The mixture was stirred at RT for 10 min. The resin bound peptide was filtered and the hydrazine treatment was repeated twice more. The resin bound peptide was washed with DMF (2 x 16 vol), DCM (2 x 16 vol) and dried with a flow of N2 over the weekend. A small aliquot of the corresponding resin bound peptide was isolated and analysed by LCMS.

Synthesis continuation
A peptide synthesizer was charged with the required amounts of 0.44 M amino acids, 1.0 M DIC, 0.5 M Oxyma, 15% piperidine/DMF solution and SPPS performed. After completion, the resin bound peptide was off-loaded, washed with DCM and dried under a flow of N2 overnight. A small aliquot of the corresponding resin bound peptide was cleaved and analyzed by LC/MS.

Example 21: Solid Phase Peptide Synthesis of Fmoc-Lys(SC)-Ala-Phe-Val-OH

DMF solutions of 0.44 M Fmoc-Phe-OH (20 mL), 1.0 M DIC (17.5 mL) and 0.5 M Oxyma (17.5 mL) were stirred in a 100 mL RBF for 25 min and added to the reaction vessel just prior to commencement of SPPS. A peptide synthesiser was charged with the required amount of 0.44M Fmoc-Ala-OH and 0.20 M Fmoc-Lys(SC)-OH/DMF solution for the coupling reactions. 2-CTC Resin previously manually loaded with Valine (3.49g, loading of 0.86 mmol/g assumed) was charged and SPPS performed. The resin bound peptide was off-loaded, washed with 100 mL of DCM and dried under a flow of N2 overnight.

Resin bound tetrapeptide (100 mg) was weighed into a HPLC vial. TIPS (30 µL), DODT (30 µL) and water (20 µL) were added. After 2 min, TFA (900 µL) was added. The mixture was shaken at RT for 1 hour. The reaction mixture was filtered using a syringe filter (20 ?m membrane), and the filtrate added to cooled MTBE (30 mL) in a centrifuge tube. The tube was sealed, and the mixture cooled in an ice bath for 20 min. The contents were split into six 5 mL Eppendorf tubes. The samples were centrifuged (8000 x g) for 10 min. The liquors were decanted, concentrated under reduced pressure and a viscous oil obtained. A few mg of the oil was dissolved in ACN/water 1:1 and analysed by LCMS. LCMS showed the desired product.
,CLAIMS:CLAIMS
We claim
1. A process for preparation of Tirzepatide or a pharmaceutically acceptable salt thereof, comprising:
(a) preparing an intermediate peptide having 19 amino acids of Formula XV,
(b) reacting the intermediate peptide having 19 amino acids with ivDde-Lys(Fmoc)-OH to form an intermediate peptide having 20 amino acids of Formula XIV

(c) deprotecting the Fmoc group of the intermediate peptide having 20 amino acids of Formula XIV to form deprotected form of the intermediate peptide of Formula XIV’

(d) reacting the intermediate peptide of Formula XIV’ with 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid to form the intermediate peptide of Formula XIII

(e) deprotecting the intermediate peptide of Formula XIII to form the intermediate peptide of Formula XIII’

(f) reacting the intermediate peptide of Formula XIII’ with 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid to form the intermediate peptide of Formula XII.

(g) deprotecting the intermediate peptide of Formula XII to form the intermediate peptide of Formula XII’

(h) reacting the intermediate peptide of Formula XII’ with Fmoc-Glu-OtBu to form an intermediate peptide of Formula XI

(i) deprotecting the intermediate peptide of Formula XI to form the intermediate peptide of Formula XI’

(j) reacting the intermediate peptide of Formula XI’ with 20-(tert-butoxy)-20-oxoicosanoic acid to form an intermediate peptide of Formula X.

(k) deprotecting the intermediate peptide of Formula X to get the intermediate peptide of Formula X’

(l) coupling remaining 19 amino acid residues in a linear manner using a solid phase peptide synthesizer to form resin bound peptide.

(m) de-blocking the resin bound peptide to form crude tirzepatide, and
(n) purifying the crude tirzepatide.
Wherein,
P is an amine protecting group such as Alloc, ivDde or Mmt;
Side chain is 2-[2-[2-[[2-[2-[2-[[5-tert-butoxy-4-[(20-tert-butoxy-20-oxo-icosanoyl)amino]-5-oxo-entanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetic acid.
2. The process according to claim 1, the protecting group P is ivDde.
3. A process for preparation of Tirzepatide or a pharmaceutically acceptable salt thereof, comprising:
(a) reacting resin bound phenyl alanine with Fmoc-Ala-OH to form resin bound dipeptide, and deprotecting the dipeptide

(b) reacting the dipeptide with Fmoc-Lys(side chain)-OH to form tripeptide,

(c) cleavage of the Fmoc protected tripeptide from the resin

(d) coupling the Fmoc protected trimer to the first resin bound peptide shown below to give an Fmoc protected version of formula X

(e) deprotection of the Fmoc group and coupling remaining amino acid residues in a linear manner using a solid phase peptide synthesizer to form resin bound 39 amino acid peptide.

(d) de-blocking the resin bound peptide to form crude tirzepatide, and
(e) purifying the crude tirzepatide.
wherein,
Side chain is 2-[2-[2-[[2-[2-[2-[[5-tert-butoxy-4-[(20-tert-butoxy-20-oxo-icosanoyl)amino]-5-oxo-entanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetic acid.
4. An intermediate peptide of Formula IV:

5. An intermediate peptide of Formula III:

6. A process for preparation of Tirzepatide or a pharmaceutically acceptable salt thereof, comprising use of at least one polypeptide or a pharmaceutically acceptable salt thereof selected from

P1 is H or Fmoc; P2 is selected from H, ivDde, Alloc and Mmt.

P1 is H or Fmoc; P2 is selected from H, ivDde, Alloc and Mmt.

P1 is H or Fmoc; P2 is selected from H, ivDde, Alloc and Mmt.

P2 is selected from H, ivDde, Alloc and Mmt.

7. An intermediate peptide of Formula XIII or a pharmaceutically acceptable salt thereof.

wherein, P1 is H or Fmoc; P2 is selected from H, ivDde, Alloc and Mmt

8. An intermediate peptide of Formula XII or a pharmaceutically acceptable salt thereof.

wherein, P1 is H or Fmoc; P2 is selected from H, ivDde, Alloc and Mmt.

9. An intermediate peptide of Formula XI or a pharmaceutically acceptable salt thereof.

wherein, P1 is H or Fmoc; P2 is selected from H, ivDde, Alloc and Mmt.

10. An intermediate peptide of Formula X or a pharmaceutically acceptable salt thereof.

wherein, P2 is selected from H, ivDde, Alloc and Mmt.