DESC:FIELD OF THE INVENTION

The present invention relates to polypeptide drugs in the field of chemical synthesis, in particular to method for synthesizing Carbetocin octapeptide.

BACKGROUND OF THE INVENTION
Carbetocin is a synthetic cyclic octapeptide containing. Carbetocin is an obstetric drug used to control postpartum hemmorage. The drug substance is chemically name is (2S)-1-[(3S, 6S, 9S, 12S, 15S)-12-[(2S)-butan-2-yl]-9-(2- carbamoylethyl)-6-(carbomoylmethyl)-15-[(4-hydroxyphenyl)methyl]-16-methyl-5, 8, 11, 14, 17- pentaoxo- 1 -thia-4,7, 10, 13, 16-pentazacycloicosane-3-carbonyl]-N-[( IS)- 1 carbamoylmethylcarbamoyl)-3- methyl-butyl] pyrrolidine-2-carboxamide and is represented by the following structural formula (I).

The structure of Carbetocin can also be represented as: Cyclo[Butyryl-Tyr(Me)-Ile-Gln-Asn-Cys]-Pro-Leu- Gly-NH; where Tyr(Me) is O-methyltyrosine, He is isoleucine, Gin is glutamine, Asn is asparagine, Cys is cysteine, Pro is proline, Leu is leucine, and Gly-N¾ is glycinamide.

Carbetocin has been approved for use immediately following an elective Cesarean section when a local or spinal anesthesia has been used. Since the uterus cannot contract on its own following incision during a Cesarean section, exogenous administration of oxytocin or an analog is necessary to restore uterine tone and prevent hemorrhage.

Carbetocin is synthetic long-acting oxytocin 8 peptide analogues with agonist properties developed by Danish Rabdosia pharmaceuticals Inc. with clinical and pharmacological properties similar to naturally occurring oxytocin like oxytocin , Carbetocin binds to oxytocin receptors of uterine smooth muscle, causing rhythmic contractions of the uterus, increasing its frequency and increasing uterine tone on the basis of the original contractions.
Solid Phase Peptide Synthesis (SPPS) of Carbetocin was first reported by M. Lebl et al. in Czech. Chem. Communications 1991, vol. 57, 1337-1344. The synthesis of Carbetocin and its analogues was reported by K. Wisniewski et al. in Journal of Medicinal Chemistry 2014, 57, 5306 - 5317. The protected carbetocin sequence was assembled using the fluorenylmethyloxycarbonyl (Fmoc)-t-butyl (t-Bu) strategy on a Rink amide resin. The couplings were mediated by diisopropylcarbodiimide (DIC)/ 1-Hydroxybenzotriazole (HOBt) using a 3-fold excess of reagents. The linear peptide was cleaved using trifluoroacetic acid (TFA)/ triisopropylsilane (TIPS)/ water (H20) cocktail, and cyclized with N,N,N',N'-Tetramethyl- 0-( lH-benzotriazol- 1-yl) uronium hexafluorophosphate, 9-(Benzotriazol-1-yl)-NN,N'N- tetramethyluronium hexafluorophosphate (HBTU)/ ?,?-Diisopropylethylamine (DIEA) in ?,?- Dimethylformamide (DMF).

Other reports of SPPS of Carbetocin by Fmoc-t-Bu strategy have appeared in CS 255616 Bl, CS 230542 B l, CN 103992390 A, CN 103833831 A, CN 103467573 A, CN 103435687 A, CN 102796178 A and CN 102260326. In all of these preceding patent publications the synthesis of Carbetocin was initiated using a Rink amide resin or Wang resin or p-Methylbenzhydrylamine (pMBHA) resin.

The prior art discloses for SPPS of Carbetocin wherein the primary amino acid sequence was sequentially assembled on the resin, and cleaved to obtain linear heptapeptide. Selective de-protection and alkylation of mercapto group of Cys with Br-(CH2)3COOC2H5 gave the heptapeptide derivative which was coupled with NBS-Tyr (Me)-OH. The resultant peptide was deprotected and cyclized with (PhO)2P(0)N3 and K2HPO4 to give Carbetocin.

The prior art also discloses for SPPS of Carbetocin protected amino acids were sequentially added followed by 4-chlorobutyric acid on resin and cleaved to obtain acylated peptide. The acylated peptide was cyclized in liquid phase under basic condition.
The prior art further discloses for SPPS of Carbetocin peptide was assembled on Rink amide resin. Protecting group was removed and cyclized on resin. The peptide was cleaved from resin, purified and lyophilized.

CN201110001400.0 discloses method with the linear Carbetocin precursor peptide of solid phase, the liquid phase cyclisation, and this method cyclisation need be at extremely rare solvent (10 -4~ 10 -5Mol/L) carry out in, produce a large amount of waste liquids, be prone to simultaneously produce polymer, cause cyclisation efficient not high, shortcomings such as after treatment complicacy.

The preparation method of the Carbetocin that application number CN201110151928.6 discloses adopts novel sulfhydryl protected amino acid Fmoc-Cys ((CH 2) 3COOAll), it is also very expensive to take off side chain allyl-based protection reagent price in this method, is unfavorable for scale operation.

European patent ES2115543 adopts a solid-liquid combined synthesis method, and the principle is as follows: firstly, obtaining 4-Cl-Butyl-Tyr (Me) -Ile-Gln-Asn-Cys (Trt) -Pro-Leu-GIy-Rink amino resin by a conventional solid phase polypeptide synthesis method, and obtaining linear 4-Cl-Butyl-Tyr (Me) -Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH after acid hydrolysis and cyclizing the linear peptide to obtain carbetocin.

Czech patent CS8605461 also adopts a solid-liquid combination synthesis method, firstly uses a solid-phase polypeptide synthesis method to synthesize Z-Ile-Gln-Asn-Cys (Bzl) -Pro-Leu-Gly-O-hydroxy resin, and then obtains Z-Ile-GIn-Asn-Cys (Bzl) -Pro-Leu-GIy-NH by cracking2Hydrogenation followed by reaction with 4-bromobutyric acid to obtain Ile-Gln-Asn-Cys ((CH)2)3COOH)-Pro-Leu-Gly-NH2 then reacting with X-Tyr (Me) -OH, deprotecting and cyclizing to obtain Carbetocin.

However, in the above-mentioned patent methods, the cyclization and cyclization are carried out in a liquid phase, which results in complicated preparation process, low cyclization yield, high preparation cost, large amount of waste liquid, and unsuitability for commercial preparation.

In view of the above, the present invention aims to provide novel methods for synthesizing Carbetocin, so that the method of the present invention significantly reduces the maximum single impurity and improves the purity of Carbetocin on the premise of ensuring the total yield of the Carbetocin.

The main object of the invention is to develop a large scale synthesis process for Carbetocin that furnishes the active pharmaceutical ingredient (API) in high yield and purity. A further object of the invention is to provide a cost effective process for the synthesis of Carbetocin.

SUMMARY OF THE INVENTION

The primary objective of the present invention is to provide a improved process for preparation of Carbetocin of formula (I)

comprising steps of:
a) Protection
b) Coupling
c) Substitution
d) Deprotection
e) Cyclization
f) Cleavage
g) Purification of Carbetocin crude
h) Lyophilization

Another objective of the present invention is to provide Carbetocin of formula (I)

comprising steps of:
a) Coupling of 4-holobutyric acid active ester of formula (III) with octapeptide-aminoresin of formula (II);

Wherein R is N-Hyroxysuccinimide; CDMT ester; Pentafluorophenol; 4-nitrophenol; 1-Hydroxypiperidine-2,6-dione
X is halo selected from chloro; Bromo
* aminoresin
in the presence of a base and solvent to obtain protected octapeptide of formula (IV)

deprotection aminoresin of protected octapeptide of formula (IV) in the presence of cleavage reagent to give linear chain of carbetocin peptide with sequence 4-chlorobutyricacid-Tyr(Me)-Ile-Gln-Asn-Cys-Pro-Leu-Gly of formula (V)

cyclization of linear peptide of formula (D) in the presence of coupling agent, base, and solvent to obtain crude Carbetocin;

purification of crude Carbetocin by preparative reversed phase high performance liquid chromatography (Prep-RP-HPLC) to obtain pure Carbetocin of formula (I).

In another aspect, the invention provides a process of preparation of intermediate intermediate 4-holobutyric acid active ester of formula (III) comprising steps of: coupling of 4-holobutyric acid of formula (V) with active ester reagent R

Wherein R is N-Hyroxysuccinimide; CDMT ester; Pentafluorophenol; 4-nitrophenol; 1-Hydroxypiperidine-2,6-dione
X is halo selected from chloro; Bromo
in the presence of a coupling agent and solvent to obtain 4-holobutyric acid active ester of formula (III).

In another aspect, the invention provides a process of preparation of intermediate octapeptide-aminoresin of formula (II), which is useful in the preparation of Carbetocin of formula (I) comprises
Coupling of aminoresin reaction with Fmoc-Gly-OH to obtain Fmoc-Gly-aminoresin inpresence of Hydroxybenzotriazole.H2O, N,N'-Diisopropylcarbodiimide, Piperidine and solvent dimethyl formamide at temperature 20-30oC to and after deprotection obtain resulting into Gly-Amino resin;

Coupling of Gly-Amino resin of formula KSM-1a with Fmoc-Leu-OH of formula KSM-2 to obtain Fmoc-Leu-Gly-Aminoresin in presence of Hydroxybenzotriazole.H2O, N,N'-Diisopropylcarbodiimide, Piperidine and solvent dimethyl formamide at temperature 20-30oC to and after deportation obtain resulting into Leu-Gly-Aminoresin of formula KSM-2a;

Coupling of Leu-Gly-Aminoresin of formula KSM-2a with Fmoc-Pro-OH of formula KSM-3 to obtain Fmoc-Pro-Leu-Gly-Aminoresin in presence of Hydroxybenzotriazole.H2O, N,N'-Diisopropylcarbodiimide, Piperidine and solvent dimethyl formamide at temperature 20-30oC to and after deportation obtain resulting into Pro-Leu-Gly-Aminoresin of formula KSM-3a;

Coupling of Pro-Leu-Gly-Aminoresin of formula KSM-3a with Fmoc-Cys(Trt)-OH of formula KSM-4 to obtain Fmoc-Cys(Trt)-Pro-Leu-Gly-Aminoresin in presence of Hydroxybenzotriazole.H2O, N,N'-Diisopropylcarbodiimide, Piperidine and solvent dimethyl formamide at temperature 20-30oC to and after deportation obtain resulting into Cys(Trt)-Pro-Leu-Gly-Aminoresin of formula KSM-4a;

Coupling of Cys(Trt)-Pro-Leu-Gly-Aminoresin of formula KSM-4a with Fmoc-Asn(Trt)-OH of formula KSM-5 to obtain Fmoc-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-Aminoresin in presence of Hydroxybenzotriazole.H2O, N,N'-Diisopropylcarbodiimide, Piperidine and solvent dimethyl formamide at temperature 20-30oC to and after deportation obtain resulting into Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-Aminoresin of formula KSM-5a;

Coupling of Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-Aminoresin of formula KSM-5a with Fmoc-Gln(Trt)-OH of formula KSM-6 to obtain Fmoc-Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-Aminoresin in presence of Hydroxybenzotriazole.H2O, N,N'-Diisopropylcarbodiimide, Piperidine and solvent dimethyl formamide at temperature 20-30oC to and after deportation obtain resulting into Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-Aminoresin of formula KSM-6a;

Coupling of Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-Aminoresin of formula KSM-6a with Fmoc-Ile-OH of formula KSM-7 to obtain Fmoc-Ile-Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-Aminoresin in presence of Hydroxybenzotriazole.H2O, N,N'-Diisopropylcarbodiimide, Piperidine and solvent dimethyl formamide at temperature 20-30oC to and after deportation obtain resulting into Ile-Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-Aminoresin of formula KSM-7a;

Coupling of Ile-Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-Aminoresin of formula KSM-7a with Fmoc-Tyr(Me)-OH of formula KSM-8 to obtain Fmoc-Tyr(Me)-Ile-Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-Aminoresin in presence of Hydroxybenzotriazole.H2O, N,N'-Diisopropylcarbodiimide, Piperidine and solvent dimethyl formamide at temperature 20-30oC to and after deportation resulting Tyr(Me)-Ile-Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-Aminoresin octapeptide-aminoresin of formula (II)

In another aspect, the invention provides a process of preparation of 4-holobutyric acid active ester of formula (III) comprises

Wherein R is N-Hyroxysuccinimide; CDMT ester; Pentafluorophenol; 4-nitrophenol; 1-Hydroxypiperidine-2,6-dione
X is halo selected from chloro; Bromo

Another objective of the present invention is to provide the synthesis technique of Carbetocin octapeptide by coupling reaction utilizing RINK AMIDE RESIN as it is an excellent tool for SPPS (Solid Phase Peptide Synthesis) of peptide amines utilizing Fmoc-protected amino acids and is commonly utilized in combinatorial chemistry to prepare amides as it can be easily cleaved by TFA (Trifluoroacetic acid).

Another objective of the present invention is to react 4-halobutyric acid with active ester reagents to synthesize the active form or active esters of above acid.

Another objective of the present invention is to synthesize Carbetocin octapeptide with >90% purity.

Another objective of the present invention is to synthesize Carbetocin octapeptide with individual impurity <0.5%.

Yet another objective of the present invention is to reduce the maximum single impurity and improve the purity of Carbetocin on the premise of ensuring the total yield of the Carbetocin.

DETAILED DESCRIPTION OF THE INVENTION

The following terms and phrases as used herein are intended to have the following meanings:
Fmoc: fluorenylmethyloxycarbonyl
Fmoc-AA: the amino acid of fluorenylmethyloxycarbonyl protection
HOBt: N-hydroxybenzotriazole
DIPC: N, N'-Diisopropylcarbodiimide
HBTU: (2-(1H-benzotriazol-1-yl)-1, 1, 3, 3-tetramethyluronium hexafluorophosphate, Hexafluorophosphate Benzotriazole Tetramethyl Uronium
THF: Tetrahydrofuran
TFA: trifluoroacetic acid
TEA: trimethylamine
DIC: N, N '-di-isopropyl carbodiimide
CDMT: 2-Chloro-4,6-dimethoxy-1,3,5-triazine
Ile: Isoleucine
Gln: glutamine
Asn: asparagine
Cys: halfcystine
Pro: proline
Leu: leucine
Gly: glycine
Trt: trityl
Me: methyl
DMF: N, N '-dimethyl formamide
Piperidine: hexahydropyridine
TFA: trifluoracetic acid
DCM: methylene dichloride
DCC: N, N '-dicyclohexylcarbodiimide

The invention provides a synthesis method of carbetocin, comprising the following steps:
a) Coupling of aminoresin with Fmoc-Gly-OH;
b) Deprotection of Fmoc;
c) Coupling comprising sequential coupling of Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH and Fmoc-Tyr(Me)-OH;
d) Deprotection to obtain octapeptide-aminoresin;
e) Coupling of 4-halobutyric acid active ester with octapeptide-aminoresin;
f) Deprotection;
g) Cyclization to obtain carbetocin crude;
h) Purification of Carbetocin crude;
i) Lyophilization.

The protecting groups are selected from Fmoc (9-Fluorenylmethoxycarbonyl), Boc (tert-Butyloxycarbonyl), Trt (Trityl), Cbz (Benzyloxycarbonyl), Alloc (Allyloxycarbonyl), Tert-Butyl, Dmb (2, 4- Dimethoxybenzyl), Fm (9-Fluorenylmethyl), Pbf (2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl), Xan (9-Xanthenyl), Tos (Tosyl), Bom (Benzyloxymethyl), Paraformaldehyde, Al (Allyl), TBDMS (tert-Butyldimethylsilyl), ONB (o- Nitrobenzyl), Meb (p-Methylbenzyl), Acm (Acetamidomethyl), Trimethylsilyl. (TMS) and Triethylsilyl (TES).

The protecting group is a protecting group which is required to protect groups interfering with synthesis such as amino, carboxyl and the like on an amino acid main chain and a side chain in the field of amino acid synthesis, and prevents the amino, the carboxyl and the like from reacting to generate impurities in the process of preparing a target product, for example, the invention protects the side chain amide groups of Asn, Cys and Gln by a Trt protecting group. Furthermore, in the protected amino acids involved in the process of the present invention, the N-terminus is preferably protected by Fmoc or Boc protecting group. Amino acids protected by a protecting group are collectively referred to as protected amino acids. Preferably, the protected Gly, protected Leu, protected Pro, and protected Cys, protected Asn, protected Gln, protected Ile and protected Tyr are as follows: Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH and Fmoc-Tyr(Me)-OH

Solvents for Solid Phase Peptide Synthesis are selected from the group of: Methylene chloride (DCM), N-methylpyrrolidone (NMP), N, N-dimethylformamide (DMF), Tetrahydrofuran (THF), Acetonitrile (CAN) and N, N-dimethylacetamide (DMA).

Coupling Reagents are selected from the group of: Dicyclohexylcarbodiimide (DCC), Diisopropylcarbodiimide (DIC), Ethyl-(N’, N’-dimethylamino)propylcarbodiimide hydrochloride (EDC), 4-(N, N-dimethylamino) pyridine (DMAP), Triethylamine (TEA), (Benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), (Benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP), (7-Azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP), Bromotripyrrolidinophosphonium hexafluorophosphate (PyBrOP), O-(Benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (HBTU), O-(Benzotriazol-1-yl)- N,N,N’,N’-tetramethyluronium tetrafluoroborate (TBTU), O-(7-Azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (HATU), O-(7-Azabenzotriazol-1-yl)- N,N,N’,N’-tetramethyluronium tetrafluoroborate (TATU), O-(6-Chlorobenzotriazol -1-yl) -N,N,N’,N’-tetramethyluronium hexafluorophosphate (HCTU), O-[(Ethoxycarbonyl) cyanomethylenamino] -N,N,N’,N’-tetramethyluronium tetrafluoro borate (TOTU), (1-Cyano-2-ethoxy-2-oxoethylidenaminooxy) dimethyl amino -morpholino- carbenium hexafluorophosphate (COMU), O-(N-Suc-cinimidyl)-1,1,3,3-tetramethyl-uronium tetrafluoroborate (TSTU), O-(5-Norbornene-2,3-dicarboximido)-N,N,N’,N’-tetramethyluronium tetrafluoroborate (TNTU), O-(1,2-Dihydro-2-oxo-1-pyridyl-N,N,N’,N’-tetramethyluronium tetrafluoroborate (TPTU), 3-(Diethylphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT), Carbonyldiimidazole (CDI), N'-Tetramethylchloroformamidinium Hexafluorophosphate (TCFH), Hydroxybenzotriazole (HOBt) and N, N’-Diisopropylcarbodiimide (DIPC).

Deprotecting groups are selected from the group consisting of: piperidine, TFA (Trifluoroacetic acid), Triisopropylsilyl (TIPS), H2 (Dihydrogen), Pd (PPh3) (triphenylphosphine) palladium, PhSiH3 (Phenylsilane), CH2Cl2 (Dichloromethane), TFMSA (Trifluoromethanesulfonic Acid), HF (Hydrogen fluoride), TBAF (Tetra-n-butylammonium fluoride), MeSiCl3 (Methyltrichlorosilane), I2 (Iodine), DTNP (2, 2'-Dithiobis (5-nitropyridine)), Tl (III) (thallium) and Hg (II) (Mercury).

Aminoresins are selected from the group of: Chlorotrityl resin, Rink amide resin, Sieber resin, PAM Resin, Wang Resin, MBHA Resin, Base Labile Resins, DHP Resin, Weinreb Aminomethyl Resin, Polyethylene Glycol-Polystyrene Grafted Resins and Amino Acid Loaded Resins.

In one of the embodiments the present invention provides novel method for synthesizing Carbetocin octapeptide by coupling reaction on the protected Gly and amino of amino resin under the action of a condensation reagent and an activation reagent to obtain peptide resin 1. Then the sequence from the C end to the N end of the Carbetocin amino acid sequence, starting from the peptide resin 1, under the action of a condensation reagent and an activation reagent, Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH and Fmoc-Tyr(Me)-OH are coupled to obtain peptide resin 2. Next, Deprotection of fmoc from the above amino acid sequence results into the N-terminal amino acid sequence.

In another aspect, the invention provides a process of preparation of intermediate octapeptide-aminoresin of formula (II), which is useful in the preparation of Carbetocin of formula (I) comprises
Coupling of aminoresin reaction with Fmoc-Gly-OH to obtain Fmoc-Gly-aminoresin inpresence of Hydroxybenzotriazole.H2O, N,N'-Diisopropylcarbodiimide, Piperidine and solvent dimethyl formamide at temperature 20-30oC to and after deprotection obtain resulting into Gly-Amino resin;

Coupling of Gly-Amino resin of formula KSM-1a with Fmoc-Leu-OH of formula KSM-2 to obtain Fmoc-Leu-Gly-Aminoresin in presence of Hydroxybenzotriazole.H2O, N,N'-Diisopropylcarbodiimide, Piperidine and solvent dimethyl formamide at temperature 20-30oC to and after deportation obtain resulting into Leu-Gly-Aminoresin of formula KSM-2a;

Coupling of Leu-Gly-Aminoresin of formula KSM-2a with Fmoc-Pro-OH of formula KSM-3 to obtain Fmoc-Pro-Leu-Gly-Aminoresin in presence of Hydroxybenzotriazole.H2O, N,N'-Diisopropylcarbodiimide, Piperidine and solvent dimethyl formamide at temperature 20-30oC to and after deportation obtain resulting into Pro-Leu-Gly-Aminoresin of formula KSM-3a;

Coupling of Pro-Leu-Gly-Aminoresin of formula KSM-3a with Fmoc-Cys(Trt)-OH of formula KSM-4 to obtain Fmoc-Cys(Trt)-Pro-Leu-Gly-Aminoresin in presence of Hydroxybenzotriazole.H2O, N,N'-Diisopropylcarbodiimide, Piperidine and solvent dimethyl formamide at temperature 20-30oC to and after deportation obtain resulting into Cys(Trt)-Pro-Leu-Gly-Aminoresin of formula KSM-4a;

Coupling of Cys(Trt)-Pro-Leu-Gly-Aminoresin of formula KSM-4a with Fmoc-Asn(Trt)-OH of formula KSM-5 to obtain Fmoc-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-Aminoresin in presence of Hydroxybenzotriazole.H2O, N,N'-Diisopropylcarbodiimide, Piperidine and solvent dimethyl formamide at temperature 20-30oC to and after deportation obtain resulting into Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-Aminoresin of formula KSM-5a;

Coupling of Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-Aminoresin of formula KSM-5a with Fmoc-Gln(Trt)-OH of formula KSM-6 to obtain Fmoc-Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-Aminoresin in presence of Hydroxybenzotriazole.H2O, N,N'-Diisopropylcarbodiimide, Piperidine and solvent dimethyl formamide at temperature 20-30oC to and after deportation obtain resulting into Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-Aminoresin of formula KSM-6a;

Coupling of Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-Aminoresin of formula KSM-6a with Fmoc-Ile-OH of formula KSM-7 to obtain Fmoc-Ile-Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-Aminoresin in presence of Hydroxybenzotriazole.H2O, N,N'-Diisopropylcarbodiimide, Piperidine and solvent dimethyl formamide at temperature 20-30oC to and after deportation obtain resulting into Ile-Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-Aminoresin of formula KSM-7a;

Coupling of Ile-Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-Aminoresin of formula KSM-7a with Fmoc-Tyr(Me)-OH of formula KSM-8 to obtain Fmoc-Tyr(Me)-Ile-Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-Aminoresin in presence of Hydroxybenzotriazole.H2O, N,N'-Diisopropylcarbodiimide, Piperidine and solvent dimethyl formamide at temperature 20-30oC to and after deportation resulting Tyr(Me)-Ile-Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-Aminoresin octapeptide-aminoresin of formula (II)

In one of the embodiments 4-halobutyric acid (halo: chloro/Bromo) is coupled into the above amino acid sequence to achieve the n-2. The 4-halobutyric acid is reacted with active ester reagents to synthesize the active form or active esters of above acid separately in another vessel. Now the isolated active esters of 4-halobutyric acid (halo: chloro/Bromo) is introduced into above amino acid sequence by maintaining the basic medium. Then intramolecular coupling reaction cyclization is carried out under the action of a condensation reagent and an activation reagent to obtain carbetocin peptide resin. Finally, Cleavage and total deprotection of peptide-aminoresin is achieved in cleavage cocktail comprising of deprotecting agent. Peptide-aminoresin is charged and reaction is maintained for 4-5 hours. This produces linear chain of carbetocin peptide with sequence 4-chlorobutyricacid-Tyr (Me)-Ile-Gln-Asn-Cys-Pro-Leu-Gly and purity 80-85%.

In another embodiment, the invention provides a process of preparation of 4-holobutyric acid active ester of formula (III) comprises

Wherein R is N-Hyroxysuccinimide; CDMT ester; Pentafluorophenol; 4-nitrophenol; 1-Hydroxypiperidine-2,6-dione
X is halo selected from chloro; Bromo

In the above embodiment process for preparation of active esters of 4-halobutyric acid as active esters comprises reaction of 4-halobutyric acid with ester reagent selected from N-Hydroxysuccinimide, CDMT, pentafluorophenol, 4-nitrophenol and 1-Hydroxypiperidine-2,6-dione to synthesize the active form or active esters of 4-halobutyric acid as active esters in the presence of coupling agent and solvent to obtain 4-holobutyric acid active ester of formula (III).

The coupling agent used in the reaction is selected from the group consisting of Benzotriazole- 1 -yl-oxy-tris- (dimethylamino)-phosphonium hexafluorophosphate (BOP), Benzotriazole- 1-yl-oxy-tris-pyrrolidino- phosphonium hexafluorophosphate (PyBOP), 0-(IH-Benzotriazol-l-yl)-N,N, N, N- tetramethyluronium tetrafluoroborate (TBTU), 0-(7-Azabenzotriazole- 1 -yl)-N,N,N,N-tetramethyluronium tetrafluoroborate (TATU), 0-(lH-Benzotriazole-l-yl)-N, N, N, N-tetramethyluronium hexafluorophosphate,(HBTU), 2-(7- Aza-lH-benzotriazole-l-yl)- 1,1, 3, 3 -tetramethyluronium hexafluorophosphate (HATU), N,N- dicyclohexylcarbodiimide (DCC), ?,?'-Diisopropylcarbodiimide (DIC) or l-Ethyl-3-(3- dimethyllaminopropyl)carbodiimide hydrochloride (EDC.HC1), preferably using EDC.HC1. The solvent used in the reaction is selected from the group consisting of ACN (acetonitrile), tetrahydrofuran (THF), ethyl acetate (EtOAc), ?,?-Dimethylformamide (DMF), N-Methylpyrrolidone (NMP), dichloromethane (DCM) or dimethylacetamide (DMAC), preferably using DMF. The reaction temperature may range from -5°C to 10°C and preferably at a temperature in the range from 0°C to 5°C. The duration of the reaction may range from 30min hour to 5 hours, preferably for a period of 1.5 hours.

The 4-halobutyric acid (halo: chloro/Bromo) is coupled with Tyr(Me)-Ile-Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-Aminoresin octapeptide-aminoresin of formula (II). It is found that the purity of octapeptide-aminoresin of formula (II) does not exceed 50% after performing the different reaction conditions like combination of HOBT and DIPC, HOBT, HBTU and DIPC which directly reduce the purity and peptide content of final molecules (n). To overcome this difficulty, 4-halobutyric acid (halo: chloro/Bromo) is reacted with active ester reagents such as N-Hydroxysuccinimide, CDMT, pentafluorophenol, 4-nitrophenol and 1-Hydroxypiperidine-2,6-dione etc to synthesize the active form or active esters of above acid separately in other vessel.

The isolated active esters of 4-halobutyric acid (halo: chloro/Bromo) is introduced into above amino acid sequence by maintaining the basic medium. List of all active esters of acid are follows and among the all active esters, the n-hydroxysuccinimide ester has given the highest purity and peptide content after analysis of final carbetocin.

The step of Coupling of 4-holobutyric acid active ester of formula (III) with octapeptide-aminoresin of formula (II);

Wherein R is N-Hyroxysuccinimide; CDMT ester; Pentafluorophenol; 4-nitrophenol; 1-Hydroxypiperidine-2,6-dione
X is halo selected from chloro; Bromo
* aminoresin
in the presence of a base and solvent to obtain protected octapeptide of formula (IV)

The base used in the reaction is selected form the group consisting of N-methylmorpholine (NMM), N, N-diisopropylethylamine (DIPEA), triethylamine, preferably using triethylamine. The solvent used in the reaction is selected from the group consisting of ACN (acetonitrile), tetrahydrofuran (THF), ethyl acetate (EtOAc), ?,?-Dimethylformamide (DMF), N-Methylpyrrolidone (NMP), dichloromethane (DCM) or dimethylacetamide (DMAC), preferably using ?,?-Dimethylformamide (DMF).

The reaction temperature may range from 5 °C to 10 °C and preferably at a temperature in the range from 0 °C to 5 °C. The duration of the reaction may range from 1 hour to 12 hours, preferably for a period of 3 hours.

Below is the schematic representation of the active esters of the given reagents:

Wherein R is N-Hyroxysuccinimide; CDMT ester; Pentafluorophenol; 4-nitrophenol; 1-Hydroxypiperidine-2,6-dione
X is halo selected from chloro; Bromo

Cleavage and total deprotection to linear chain of carbetocin peptide:

The peptide-aminoresin is washed with solvents. The deprotection of protected octapeptide of formula (IV) in the presence of cleavage reagent to obtain linear peptide of formula (V), and dried under vacuum. Cleavage and total deprotection of peptide-aminoresin is achieved in cleavage cocktail comprising of TFA/TIPS/Water/Phenol. This produces linear chain of carbetocin peptide with sequence 4-chlorobutyricacid-Tyr(Me)-Ile-Gln-Asn-Cys-Pro-Leu-Gly of formula (V).
The cleavage reagent containing the mixture of TFA (Trifluoroacetic acid), TIPS (Triisopropylsilane), water and Phenol; the ratio is 36.8: 2 : 1 : 1, preferably using TFA 155.6 ml, TIPS 8.6 ml, Water 4.3 ml and Phenol 4.3 ml. The solvent used in the reaction is selected from the group consisting of DCM, diisopropyl ether, THF, DMF, NMP or DMAc, preferably using DCM and diisopropyl ether. The reaction temperature may range from below 0 to 5°C.
Cyclization of linear 4-chlorobutyricacid-Tyr(Me)-Ile-Gln-Asn-Cys-Pro-Leu-Gly of formula (V) to obtain crude Carbetocin:

The cyclization of linear 4-chlorobutyricacid-Tyr(Me)-Ile-Gln-Asn-Cys-Pro-Leu-Gly of formula (V) in the presence of base and solvent to obtain crude Carbetocin, at tempreture below -40?. Further preferably the base is as aqueous solution of ammonia which was charged slowly by maintaining the reaction at below -40?. Slowly, the reaction temperature is elevated to 55? and maintained for 2-2.5 hours. The reaction is completed when linear carbetocin peptide remains less than 2%.
The base used in the reaction is selected form the group consisting of aqueous solution of ammonia, NMM, DIPEA, TEA or collidine, preferably using aqueous solution of ammonia. The solvent used in the reaction is selected from the group consisting of THF, DMF, NMP or DMAc, preferably using DMF. The reaction temperature may range from below -40°C and preferably at a temperature in the range from -30°C to -45°C.
Purification of Carbetocin crude:
In an embodiment the linear carbetocin peptide is dissolved in a solvent and the solution is cooled. An aqueous solution of ammonia is charged slowly by maintaining the reaction at certain temperature. Slowly, the reaction temperature is elevated and maintained for few of hours. The reaction is completed when linear carbetocin peptide remains less than 2% as monitored using analytical technique i.e. HPLC. Carbetocin crude is purified using preparative HPLC. The eluted Carbetocin is collected, analyzed and lyophilized.

Carbetocin crude is purified using preparative HPLC. Preparative column packed with 5µm C18 silica is first saturated with ammonium acetate buffer. Carbetocin crude is dissolved in water and injected. The column is washed with water. A gradient of ammonium acetate buffer and acetonitrile is used as mobile phase. Carbetocin is eluted at 60-70 minutes. Fractions are collected, analyzed and lyophilized. The obtained carbetocin is >99% pure with any individual impurity <0.5%.

Reaction scheme:

Examples
Below in conjunction with embodiments, the invention will be further described:

Example 1: Preparation of aminoresin

Rink amide aminoresin with loading capacity 0.7-1.2 mmole/g is swelled in dimethyl formamide (DMF) for 30 minutes.

The coupling amino resin with first Fmoc protected amino acid Fmoc-Gly-OH 4.2g, HOBt.H2O 2.1g, and DIC 2.2ml is dissolved in DMF on ice bath and charged slowly in reaction vessel to control the reaction temperature below 25?. Coupling reaction is continued for 1.5-2 hours and reaction completion is ascertained by ninhydrin reagent. The degree of substitution for Fmoc-Gly-OH is in the range of 0.6-0.65 mmole/g, resulting into Fmoc-Gly-Amino resin.

Subsequently, Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH and Fmoc-Tyr(Me)-OH are coupled using HOBt.H2O/ DIPC in DMF. The resulting sequence is Fmoc-Tyr(Me)-Ile-Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-Aminoresin. Deprotection of fmoc from the above amino acid sequence results into the N-terminal amino acid sequence.

Example 2: Synthesis of 4-chlorobutyric acid - Hydroxysuccinimide active ester:
4-Chlorobutyric acid 5.3g is dissolved in THF at 0-5oC. N-Hydroxysuccinimide 5.6g and N,N'-Dicyclohexylcarbodiimide (DCC) 6.8g is added slowly and reacted for 1 hour. Reaction mass is filtered and the filtrate is distilled. Active ester is precipitated in ethyl acetate.

Example 3: Synthesis of 4-chlorobutyric acid - 2-Chloro-4,6-dimethoxy-1,3,5-triazineactive ester:
4-Chlorobutyric acid 6.3g is dissolved in THF at 0-5oC. 2-Chloro-4,6-dimethoxy-1,3,5-triazine 6.9g and N,N'-Dicyclohexylcarbodiimide (DCC) 7.2g is added slowly and reacted for 1 hour. Reaction mass is filtered and the filtrate is distilled. Active ester is precipitated in ethyl acetate.
Example 4: Synthesis of 4-bromobutyric acid -pentafluorophenol active ester:
4-bromobutyric acid 7.1g is dissolved in THF at 0-5oC. pentafluorophenol 5.9g and N,N'-Dicyclohexylcarbodiimide (DCC) 6.2g is added slowly and reacted for 1 hour. Reaction mass is filtered and the filtrate is distilled. Active ester is precipitated in ethyl acetate.
Example 5: Synthesis of 4-chlorobutyric acid -4-nitrophenol active ester:
4-chlorobutyric acid 5.5g is dissolved in THF at 0-5oC. pentafluorophenol 5.4g and N,N'-Dicyclohexylcarbodiimide (DCC) 6.9g is added slowly and reacted for 1 hour. Reaction mass is filtered and the filtrate is distilled. Active ester is precipitated in ethyl acetate.
Example 6: Synthesis of 4-chlorobutyric acid -1-Hydroxypiperidine-2,6-dione active ester:
4-chlorobutyric acid 5.6g is dissolved in THF at 0-5oC. 1-Hydroxypiperidine-2,6-dione 5.2g and N,N'-Dicyclohexylcarbodiimide (DCC) 6.3g is added slowly and reacted for 1 hour. Reaction mass is filtered and the filtrate is distilled. Active ester is precipitated in ethyl acetate.
The active ester synthesized is of the following formula:

Where, ‘R’ is selected from the group of N-Hydroxysuccinimide, CDMT, pentafluorophenol, 4-nitrophenol and 1-Hydroxypiperidine-2,6-dione.

Example 7: Coupling of 4-chlorobutyric acid active ester with octapeptide-aminoresin:

4-Chlorobutyric acid - N-Hydroxysuccinimide active ester 4.95g, triethylamine(TEA) 2.3 ml is dissolved in DMF on ice bath and charged slowly in reaction vessel to control the reaction temperature below 25oC. Resin is stirred for 3 hours and reaction completion is ascertained by ninhydrin reagent. The resulting sequence is 4-chlorobutyricacid-Tyr(Me)-Ile-Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-Aminoresin.

Example 8: Cleavage and total Deprotection:

The peptide-aminoresin is washed with DCM and diisopropyl ether and dried under vacuum. Cleavage and total deprotection of peptide-aminoresin is achieved in cleavage cocktail comprising of TFA/TIPS/Water/Phenol. TFA 155.6 ml, TIPS 8.6 ml, Water 4.3 ml and Phenol 4.3 ml is cooled on ice bath to 0-5oC. Peptide-aminoresin is charged and reaction is maintained for 4-5 hours. This produces linear chain of carbetocin peptide with sequence 4-chlorobutyricacid-Tyr(Me)-Ile-Gln-Asn-Cys-Pro-Leu-Gly and purity 93.6%.

Example 9: Cyclization
The peptide-aminoresin is washed with DCM and diisopropyl ether and dried under vacuum. Cleavage and total deprotection of peptide-aminoresin is achieved in cleavage cocktail comprising of TFA/TIPS/Water/Phenol. TFA 155.6 ml, TIPS 8.6 ml, Water 4.3 ml and Phenol 4.3 ml is cooled on ice bath to 0-5oC. Peptide-aminoresin is charged and reaction is maintained for 4-5 hours. This produces linear chain of carbetocin peptide with sequence 4-chlorobutyricacid-Tyr(Me)-Ile-Gln-Asn-Cys-Pro-Leu-Gly and purity 95.2% with peptide content 60-65%.

Example 10: Purification of Carbetocin crude:

Carbetocin crude is purified using preparative HPLC. Preparative column packed with 5µm C18 silica is first saturated with ammonium acetate buffer. Carbetocin crude is dissolved in water and injected. The column is washed with water. A gradient of ammonium acetate buffer and acetonitrile is used as mobile phase. Carbetocin is eluted at 60-70 minutes. Fractions are collected, analyzed and lyophilized. The obtained carbetocin is 99.5% pure with any individual impurity <0.5%.

The 4-halobutyric acid (halo: chloro/Bromo) is coupled with Tyr(Me)-Ile-Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-Aminoresin octapeptide-aminoresin of formula (II). It is found that the purity of octapeptide-aminoresin of formula (II) does not exceed 50% after performing the different reaction conditions like combination of HOBT and DIPC, HOBT, HBTU and DIPC which directly reduce the purity and peptide content of final molecules (n). To overcome this difficulty, 4-halobutyric acid (halo: chloro/Bromo) is reacted with active ester reagents such as N-Hydroxysuccinimide, CDMT, pentafluorophenol, 4-nitrophenol and 1-Hydroxypiperidine-2,6-dione etc to synthesize the active form or active esters of above acid separately in other vessel.

Coupling with octapeptide-aminoresin of formula (II) Purity of linear chain of carbetocin peptide Purity of Carbetocin crude Purity of Carbetocin after Purification
4-chlorobutyric acid - Hydroxysuccinimide active ester 93.6% 95.2% 99.5%
4-chlorobutyric acid - 2-Chloro-4,6-dimethoxy-1,3,5-triazineactive ester 91.8% 94.7% 99.2%
4-bromobutyric acid -pentafluorophenol active ester 91.0% 94.0% 99.4%
4-chlorobutyric acid -4-nitrophenol active ester 92.2% 93.8% 99.2%
4-chlorobutyric acid -1-Hydroxypiperidine-2,6-dione active ester 92.0% 94.1% 99.5%
4-chlorobutyric acid coupling in presences of HOBT and DIPC 48.0% 63.2% 98.5%
4-chlorobutyric acid coupling in presences of HOBT, HBTU and DIPC 51.2% 66.5% 98.5%

,CLAIMS:CLAMIS
We Claim,
1. A improved process for preparation of Carbetocin of formula (I)

comprising steps of:
b) Coupling of 4-holobutyric acid active ester of formula (III) with octapeptide-aminoresin of formula (II);

Wherein R is N-Hyroxysuccinimide; CDMT ester; Pentafluorophenol; 4-nitrophenol; 1-Hydroxypiperidine-2,6-dione
X is halo selected from chloro; Bromo
* aminoresin
in the presence of a base and solvent to obtain protected octapeptide of formula (IV);
c) deprotection aminoresin of protected octapeptide of formula (IV) in the presence of cleavage reagent to give linear chain of carbetocin peptide with sequence 4-chlorobutyricacid-Tyr(Me)-Ile-Gln-Asn-Cys-Pro-Leu-Gly of formula (V)

d) cyclization of linear peptide of formula (D) in the presence of base and solvent to obtain crude Carbetocin;

e) purification of crude Carbetocin by preparative reversed phase high performance liquid chromatography (Prep-RP-HPLC) to obtain pure Carbetocin of formula (I).

2. The improved process for preparation of Carbetocin as claimed in claim 1 wherein in step(a) base selected from N-methylmorpholine (NMM), N, N-diisopropylethylamine (DIPEA), triethylamine, preferably using triethylamine and solvent selected from the group consisting of ACN (acetonitrile), tetrahydrofuran (THF), ethyl acetate (EtOAc), ?,?-Dimethylformamide (DMF), N-Methylpyrrolidone (NMP), dichloromethane (DCM) or dimethylacetamide (DMAC), preferably using ?,?-Dimethylformamide (DMF)

3. The improved process for preparation of Carbetocin as claimed in claim 1wherein in step(b) cleavage reagent containing the mixture of TFA (Trifluoroacetic acid), TIPS (Triisopropylsilane), water and Phenol in the ratio is 36.8: 2 : 1 : 1and solvent is selected from DCM, diisopropyl ether, THF, DMF, NMP or DMAc, preferably using DCM and diisopropyl ether.

4. The improved process for preparation of Carbetocin as claimed in claim 1wherein in step(c) base is selected form the group consisting of aqueous solution of ammonia, NMM, DIPEA, TEA or collidine, and solvent is selected from the group consisting of THF, DMF, NMP or DMAc, preferably using DMF.

5. The improved process for preparation of Carbetocin as claimed in claim 1 process of preparation of intermediate 4-holobutyric acid active ester of formula (III) comprises
comprising steps of:
Coupling of 4-holobutyric acid of formula (V) with active ester reagent R

Wherein R is N-Hyroxysuccinimide; CDMT ester; Pentafluorophenol; 4-nitrophenol; 1-Hydroxypiperidine-2,6-dione
X is halo selected from chloro; Bromo
in the presence of a coupling agent and solvent to obtain 4-holobutyric acid active ester of formula (III).

6. The improved process for preparation of Carbetocin as claimed in claim 5 wherein solvent is selected from the group consisting of ACN (acetonitrile), tetrahydrofuran (THF), ethyl acetate (EtOAc), ?,?-Dimethylformamide (DMF), N-Methylpyrrolidone (NMP), dichloromethane (DCM) or dimethylacetamide (DMAC).

7. The improved process for preparation of Carbetocin as claimed in claim 5 wherein coupling agent used in the reaction is selected from the group consisting of Benzotriazole- 1 -yl-oxy-tris- (dimethylamino)-phosphonium hexafluorophosphate (BOP), Benzotriazole- 1-yl-oxy-tris-pyrrolidino- phosphonium hexafluorophosphate (PyBOP), 0-(IH-Benzotriazol-l-yl)-N,N, N, N- tetramethyluronium tetrafluoroborate (TBTU), 0-(7-Azabenzotriazole- 1 -yl)-N,N,N,N-tetramethyluronium tetrafluoroborate (TATU), 0-(lH-Benzotriazole-l-yl)-N, N, N, N-tetramethyluronium hexafluorophosphate,(HBTU), 2-(7- Aza-lH-benzotriazole-l-yl)- 1,1, 3, 3 -tetramethyluronium hexafluorophosphate (HATU), N,N- dicyclohexylcarbodiimide (DCC), ?,?'-Diisopropylcarbodiimide (DIC) or l-Ethyl-3-(3- dimethyllaminopropyl)carbodiimide hydrochloride (EDC.HC1).

8. The improved process for preparation of Carbetocin as claimed in claim 1 process of preparation of intermediate octapeptide-aminoresin of formula (II), which is useful in the preparation of Carbetocin of formula (I) comprises
a) Coupling of aminoresin reaction with Fmoc-Gly-OH to obtain Fmoc-Gly-aminoresin inpresence of Hydroxybenzotriazole.H2O, N,N'-Diisopropylcarbodiimide, Piperidine and solvent dimethyl formamide at temperature 20-30oC to and after deprotection obtain resulting into Gly-Amino resin;

b) Coupling of Gly-Amino resin of formula KSM-1a with Fmoc-Leu-OH of formula KSM-2 to obtain Fmoc-Leu-Gly-Aminoresin in presence of Hydroxybenzotriazole.H2O, N,N'-Diisopropylcarbodiimide, Piperidine and solvent dimethyl formamide at temperature 20-30oC to and after deportation obtain resulting into Leu-Gly-Aminoresin of formula KSM-2a;

c) Coupling of Leu-Gly-Aminoresin of formula KSM-2a with Fmoc-Pro-OH of formula KSM-3 to obtain Fmoc-Pro-Leu-Gly-Aminoresin in presence of Hydroxybenzotriazole.H2O, N,N'-Diisopropylcarbodiimide, Piperidine and solvent dimethyl formamide at temperature 20-30oC to and after deportation obtain resulting into Pro-Leu-Gly-Aminoresin of formula KSM-3a;

d) Coupling of Pro-Leu-Gly-Aminoresin of formula KSM-3a with Fmoc-Cys(Trt)-OH of formula KSM-4 to obtain Fmoc-Cys(Trt)-Pro-Leu-Gly-Aminoresin in presence of Hydroxybenzotriazole.H2O, N,N'-Diisopropylcarbodiimide, Piperidine and solvent dimethyl formamide at temperature 20-30oC to and after deportation obtain resulting into Cys(Trt)-Pro-Leu-Gly-Aminoresin of formula KSM-4a;

e) Coupling of Cys(Trt)-Pro-Leu-Gly-Aminoresin of formula KSM-4a with Fmoc-Asn(Trt)-OH of formula KSM-5 to obtain Fmoc-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-Aminoresin in presence of Hydroxybenzotriazole.H2O, N,N'-Diisopropylcarbodiimide, Piperidine and solvent dimethyl formamide at temperature 20-30oC to and after deportation obtain resulting into Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-Aminoresin of formula KSM-5a;

f) Coupling of Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-Aminoresin of formula KSM-5a with Fmoc-Gln(Trt)-OH of formula KSM-6 to obtain Fmoc-Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-Aminoresin in presence of Hydroxybenzotriazole.H2O, N,N'-Diisopropylcarbodiimide, Piperidine and solvent dimethyl formamide at temperature 20-30oC to and after deportation obtain resulting into Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-Aminoresin of formula KSM-6a;

g) Coupling of Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-Aminoresin of formula KSM-6a with Fmoc-Ile-OH of formula KSM-7 to obtain Fmoc-Ile-Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-Aminoresin in presence of Hydroxybenzotriazole.H2O, N,N'-Diisopropylcarbodiimide, Piperidine and solvent dimethyl formamide at temperature 20-30oC to and after deportation obtain resulting into Ile-Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-Aminoresin of formula KSM-7a;

h) Coupling of Ile-Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-Aminoresin of formula KSM-7a with Fmoc-Tyr(Me)-OH of formula KSM-8 to obtain Fmoc-Tyr(Me)-Ile-Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-Aminoresin in presence of Hydroxybenzotriazole.H2O, N,N'-Diisopropylcarbodiimide, Piperidine and solvent dimethyl formamide at temperature 20-30oC to and after deportation resulting Tyr(Me)-Ile-Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-Aminoresin octapeptide-aminoresin of formula (II)

9. The improved process for preparation of Carbetocin as claimed in claim 1 wherein synthesis technique of Carbetocin octapeptide by coupling reaction utilizing RINK AMIDE RESIN as it is an excellent tool for SPPS (Solid Phase Peptide Synthesis) of peptide amines utilizing Fmoc-protected amino acids and is commonly utilized in combinatorial chemistry to prepare amides as it can be easily cleaved by TFA (Trifluoroacetic acid).

Dated this 22nd Day of March 2022