DESC:RELATED PATENT APPLICATION:

This application claims the priority to and benefit of Indian Patent Application No. 202241053596 filed on September 19, 2022; the disclosures of which are incorporated herein by reference.

FIELD OF THE INVENTION:

The present invention relates to an improved process for the preparation of Difelikefalin Acetate having the chemical Formula I

Formula I
The present invention also relates to novel intermediate compounds of Formula IIIb, Formula IVb, Formula Vb and Fragment 2, and their preparation processes.

wherein, R2 and R3 is selected from carbonyl, amine, alkoxide, Boc protecting group or Fmoc protecting group; R2 and R3 can be bonded together to form a carbocyclic or heterocyclic, saturated, or unsaturated ring, which may optionally be mono- or poly-substituted with keto, hydroxy, amino, halogens, radicals containing N, O, P atoms; R2’ and R4 is amine protecting group. The present invention further relates to use of said novel intermediates in the preparation of Difelikefalin Acetate.

BACKGROUND OF THE INVENTION:

DIFELIKEFALIN ACETATE (KORSUVA) is an analgesic opioid peptide used for the treatment of moderate-to-severe pruritus. It acts as a peripherally specific, highly selective agonist of the ?-opioid receptor (KOR).

Difelikefalin was approved for medical use in the United States in August 2021, sold under the brand name KORSUVA.

Difelikefalin and its process for the preparation was first disclosed in US 7,402,564 B1. In this process, there is a possibility of formation of several impurities which shows impact on yield as well as purity of final API, and additional purification techniques are also required to obtain pure Difelikefalin.

In view of all these disadvantages, there is a significant need to develop a cost effective, stable, commercially viable, large scale and robust process for the preparation of highly pure Difelikefalin Acetate with good yield.

OBJECTS OF THE INVENTION:

The primary object of the present invention is to provide an efficient and industry feasible process for the preparation of Difelikefalin Acetate.

Another object of the present invention is to provide an improved process for the preparation of highly pure Difelikefalin Acetate with good yield.

Yet another object of the present invention is to provide an improved process for preparation of Difelikefalin Acetate via formation of novel intermediates.
SUMMARY OF THE INVENTION:

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

In one aspect present invention discloses an improved process for the preparation of Difelikefalin Acetate having the structural Formula I, by using solution phase synthesis.

The said process comprises the steps of:
i. coupling of Fragment-1 with Fragment-2 in presence of coupling reagent to obtain compound of Formula Ic;

wherein R2 and R3 is selected from the group consisting of carbonyl, amine, alkoxide, Boc protecting group or Fmoc protecting group; R2 and R3 can be bonded together to form a carbocyclic or heterocyclic, saturated or unsaturated ring, which may optionally be mono- or poly-substituted with keto, hydroxy, amino, halogens, radicals containing N, O, P atoms; R4 is amine protecting group selected from Fmoc, Boc or Cbz;
ii. deprotection of compound of Formula Ic in presence of a base to obtain compound of Formula IIc;

optionally, compound of Formula IIc after base treatment the obtained deprotected compound reacts again with amine protecting group to obtain amine protected compound;
iii. deprotection of compound of Formula IIc with TFA or HCl to obtain compound of Formula IId as TFA salt or HCl salt; and

iv. purification of compound of Formula IId in presence of Acetic acid or Ammonium acetate to obtain pure Difelikefalin Acetate of compound of Formula I.

In another aspect, present invention discloses an improved process for the preparation of Difelikefalin Acetate novel intermediate (Fragment 2) by using solution phase synthesis.

The said process comprises the steps of:
a) coupling of compound of Formula Ib with compound of Formula IIb in presence of coupling reagent to obtain compound of Formula IIIb;

wherein, R2 and R3 is selected from carbonyl, amine, alkoxide, Boc protecting group or Fmoc protecting group or bonded together to form a carbocyclic or heterocyclic, saturated or unsaturated ring, which may optionally be mono- or poly-substituted with keto, hydroxy, amino, atoms containing N, O; R2’ and R4 is amine protecting group selected from Fmoc, Boc or Cbz;
b) deprotection of compound of Formula IIIb to obtain compound of Formula IVb;

c) reacting compound of Formula IVb with amine protected leucine compound to obtain compound of Formula Vb; and

d) deprotection of compound of Formula Vb to obtain compound of Fragment 2.

In another aspect present invention discloses a process for the preparation of compound of Formula IIIb

which comprises coupling of compound of Formula Ib with compound of Formula IIb in presence of coupling reagent to obtain compound of Formula IIIb

wherein, R2 and R3 is selected from carbonyl, amine, alkoxide, Boc protecting group or Fmoc protecting group or bonded together to form a carbocyclic or heterocyclic, saturated, or unsaturated ring, which may optionally be mono- or poly-substituted with keto, hydroxy, amino, atoms containing N, O; R2’ and R4 is amine protecting group selected from Fmoc, Boc or Cbz.

In one more aspect, present invention discloses a process for the preparation of compound of Formula IVb

which comprises deprotection of compound of Formula IIIb to obtain compound of Formula IVb

wherein, R2 and R3 is selected from carbonyl, amine, alkoxide, Boc protecting group or Fmoc protecting group or bonded together to form a carbocyclic or heterocyclic, saturated or unsaturated ring, which may optionally be mono- or poly-substituted with keto, hydroxy, amino, atoms containing N, O; R2’ and R4 is amine protecting group selected from Fmoc, Boc or Cbz.
In further aspect present invention discloses a process for the preparation of compound of Formula Vb

which comprises reaction of compound of Formula IVb with amine protected leucine compound to obtain compound of Formula Vb

wherein, R2 and R3 is selected from carbonyl, amine, alkoxide, Boc protecting group or Fmoc protecting group or bonded together to form a carbocyclic or heterocyclic, saturated or unsaturated ring, which may optionally be mono- or poly-substituted with keto, hydroxy, amino, atoms containing N, O; R2’ and R4 is amine protecting group selected from Fmoc, Boc or Cbz.

In another aspect present invention provides an improved process for the preparation of Difelikefalin Acetate intermediate (Fragment-1) by using solution phase synthesis.

The said process comprises the steps of:
a) coupling of compound of Formula Ia with N-Hydroxysuccinimide (NHS) in presence of reagent to obtain compound of Formula IIa;

wherein, R1 is selected from benzyl, alkyl, Boc protecting group or Fmoc protecting group;
b) coupling of compound of Formula IIa with D-Phe-OH in presence of base to obtain Fragment-1.

In the said process, coupling reagent is selected from the group consisting of N,N'-Diisopropylcarbodiimide (DIC), Dicyclohexyl carbodiimide (DCC), 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).HCl, 1-Hydroxybenzotriazole (HOBT), 1-Hydroxy-7-azabenzotriazole (HOAT), and mixture thereof.

The base employed in said process is selected from the group consisting of potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, lithium hydroxide, ammonium hydroxide, di-isopropyl amine, N, N-di-isopropyl ethylamine, triethylamine, tertiary butyl amine, piperidine, diethylamine, piperidine in dimethyl formamide and mixture thereof.

The said process employs solvent selected from the group consisting of methanol, ethanol, isopropanol, chloroform, methylene chloride, ethyl acetate, isopropyl acetate, diethyl ether, tetrahydrofuran (THF), 2-methyltetrahydrofuran, diisopropyl ether (DIPE), Methyl tert-butyl ether (MTBE), acetonitrile, dimethylformamide, dimethylacetamide, 1,4-dioxane, N-Methyl-2-pyrrolidone (NMP), acetone, hexane, heptane, water, and mixture thereof.

Yet in another aspect present invention provides novel intermediate compounds as shown below:

wherein, R2 and R3 is selected from carbonyl, amine, alkoxide, Boc protecting group or Fmoc protecting group or bonded together to form a carbocyclic or heterocyclic, saturated or unsaturated ring, which may optionally be mono- or poly-substituted with keto, hydroxy, amino, atoms containing N, O; R2’ and R4 is amine protecting group selected from Fmoc, Boc or Cbz.

DETAILED DESCRIPTION OF THE INVENTION:

The present invention provides a process for the preparation of Difelikefalin Acetate by making appropriate fragments, followed by condensing these fragments using solution phase approach with higher yields and purity.

Unless otherwise stated, the following terms used in the specification have the meanings given below:

Solvents used throughout the invention is selected from the group consisting of alcoholic solvents such as methanol, ethanol, isopropanol; chlorinated solvents such as chloroform, methylene chloride; ester solvents such as ethyl acetate, butyl acetate, isopropyl acetate; ether solvents such as diethyl ether, tetrahydrofuran (THF), 2-methyltetrahydrofuran, diisopropyl ether (DIPE), Methyl tert-butyl ether (MTBE); nitrile solvents such as acetonitrile; aprotic solvents and protic solvents such as dimethylformamide, dimethyl sulfoxide (DMSO), dimethylacetamide, 1,4-dioxane, N-Methyl-2-pyrrolidone (NMP), acetone, hexane, heptane, water and mixture thereof.

Coupling reagents used throughout the invention is selected from the group consisting of N,N'-Diisopropylcarbodiimide (DIC), Dicyclohexyl carbodiimide (DCC), 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).HCl, 1-Hydroxybenzotriazole (HOBT), 1-Hydroxy-7-azabenzotriazole (HOAT) and mixture thereof.

Additives used throughout the invention is selected from the group consisting of ethyl 2-cyano-2-(hydroxyimino)acetate (oxyma).

Coupling of amino acid is carried out in presence of a base or without using a base. The base is organic or inorganic base. The inorganic base is selected from the group consisting of potassium carbonate, lithium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, sodium hydroxide, potassium hydroxide, lithium hydroxide, ammonium hydroxide and mixture thereof; the organic base is selected from the group consisting of diisopropyl amine, N,N-diisopropyl ethylamine, triethylamine, tertiary butyl amine, dimethylamine, tri methyl amine, isopropyl ethylamine, pyridine, N-methyl morpholine (NMM) and mixture thereof.

Base used in the deprotection throughout the invention is selected from group consisting of diethylamine, tert-butyl amine, pyridine, 4-methyl piperidine, 4-methyl piperidine in dimethyl formamide, piperidine in dimethyl formamide and piperazine in dimethyl formamide, N-methyl morpholine (NMM) and mixture thereof.

Reagent used for deprotection used throughout the invention is preferably palladium on carbon.

Buffer used for deprotection used throughout the invention is ammonium acetate or sodium acetate.

Scavenger is used for deprotection throughout the invention is selected from the group consisting of Triisopropyl silane (TIPS), Thioanisole, phenol, water, DithioThreitol (DTT) or 3,6-dioxa-1,8-octanedithiol (DODT).

Amine protected leucine compound is Fmoc-D-Leu-OH.

Accordingly, the present invention provides an improved process for the preparation of Difelikefalin Acetate having the sequence chemical Formula (I).

Formula I

In one aspect present invention provides an improved process for the preparation of Difelikefalin Acetate, which is outlined below in Scheme I:

Scheme I
wherein, R1 is selected from benzyl, alkyl, Boc protecting group or Fmoc protecting group; R2 and R3 is selected from carbonyl, amine, alkoxide, Boc protecting group or Fmoc protecting group or bonded together to form a carbocyclic or heterocyclic, saturated or unsaturated ring, which may optionally be mono- or poly-substituted with keto, hydroxy, amino, atoms containing N, O; R4 is amine protecting group selected from Fmoc, Boc or Cbz.

In stage-Ic), reacting compound of Fragment-1 with compound of Fragment-2 in presence of suitable coupling reagent to obtain compound of Formula Ic;

In stage-IIc), deprotecting the compound of Formula Ic in presence of suitable base to obtain compound of Formula IIc;

In stage-IIIc), deprotection of the compound of Formula IIc followed by reaction with Trifluoroacetic acid (TFA) or Hydrochloric acid to obtain compound of Formula IId;

In stage-IVc), reacting compound of Formula IId with acetic acid or Ammonium acetate followed by purification by lyophilization to obtain compound of pure Difelikefalin Acetate salt (Formula I).

In another aspect, the present invention provides an improved process for the preparation of compound of Fragment 2 by using novel intermediate compounds, which is outlined below in Scheme-II:

Scheme II
wherein, R2 and R3 is selected from carbonyl, amine, alkoxide, Boc protecting group or Fmoc protecting group or bonded together to form a carbocyclic or heterocyclic, saturated or unsaturated ring, which may optionally be mono- or poly-substituted with keto, hydroxy, amino, atoms containing N, O; R2’ and R4 is amine protecting group selected from Fmoc, Boc or Cbz.

In stage-Ib), coupling of compound of Formula Ib with compound of Formula IIb in presence of suitable coupling reagent to obtain novel intermediate compound of Formula IIIb;

In stage-IIb), deprotection of compound of Formula IIIb to obtain novel intermediate compound of Formula IVb;

In stage-IIIb), reacting of compound of Formula IVb with suitable amine protected leucine compound to obtain novel intermediate compound of Formula Vb;

In stage-IVb), deprotection of compound of Formula Vb to obtain novel intermediate compound of Fragment 2.

In another aspect, the present invention provides an improved process for the preparation of compound of Fragment 1, which is outlined below in Scheme-III:

Scheme III
wherein, R1 is selected from benzyl, alkyl, Boc protecting group or Fmoc protecting group.

In stage-Ia), coupling of compound of Formula Ia with N-Hydroxysuccinimide (NHS) in presence of suitable coupling reagent to obtain compound of Formula IIa;

In stage-IIa), coupling of compound of Formula IIa with D-Phe-OH in presence of suitable base to obtain Fragment 1.

In another aspect, the present invention provides process for the preparation of novel intermediate compound of Formula IIIb, which is outlined below in Scheme-IV:

Scheme IV
wherein, R2 and R3 is selected from carbonyl, amine, alkoxide, Boc protecting group or Fmoc protecting group or bonded together to form a carbocyclic or heterocyclic, saturated or unsaturated ring, which may optionally be mono- or poly-substituted with keto, hydroxy, amino, atoms containing N, O; R2’ and R4 is amine protecting group selected from Fmoc, Boc or Cbz.

In above stage, coupling with compound of Formula Ib with compound of Formula IIb in presence of coupling reagent to obtain novel intermediate compound of Formula IIIb.

In another aspect, the present invention provides process for the preparation of novel intermediate compound of Formula IVb which is outlined below in Scheme-V:

Scheme V
wherein, R2 and R3 is selected from carbonyl, amine, alkoxide, Boc protecting group or Fmoc protecting group or bonded together to form a carbocyclic or heterocyclic, saturated or unsaturated ring, which may optionally be mono- or poly-substituted with keto, hydroxy, amino, atoms containing N, O; R2’ and R4 is amine protecting group selected from Fmoc, Boc or Cbz.

In above stage, deprotection of compound of Formula IIIb to obtain novel intermediate compound of Formula IVb.

In another aspect, the present invention provides process for the preparation of novel intermediate compound of Formula Vb, which is outlined below in Scheme-VI:

Scheme VI
wherein, R2 and R3 is selected from carbonyl, amine, alkoxide, Boc protecting group or Fmoc protecting group or bonded together to form a carbocyclic or heterocyclic, saturated or unsaturated ring, which may optionally be mono- or poly-substituted with keto, hydroxy, amino, atoms containing N, O; R4 is amine protecting group selected from Fmoc, Boc or Cbz.

In above stage, reacting of compound of Formula IVb with amine protected leucine compound to obtain novel intermediate compound of Formula Vb.

In one aspect present invention also discloses an intermediate compound of formula IIIb

wherein, R2 and R3 is selected from carbonyl, amine, alkoxide, Boc protecting group or Fmoc protecting group; R2 and R3 can be bonded together to form a carbocyclic or heterocyclic, saturated or unsaturated ring, which may optionally be mono- or poly-substituted with keto, hydroxy, amino, halogens, radicals containing N, O, P atoms; R2’ and R4 is amine protecting group selected from Fmoc, Boc or Cbz.

In another aspect present invention discloses an intermediate compound of formula IVb

wherein, R2 and R3 is selected from carbonyl, amine, alkoxide, Boc protecting group or Fmoc protecting group; R2 and R3 can be bonded together to form a carbocyclic or heterocyclic, saturated or unsaturated ring, which may optionally be mono- or poly-substituted with keto, hydroxy, amino, halogens, radicals containing N, O, P atoms; R4 is amine protecting group selected from Fmoc, Boc or Cbz.

In another aspect present invention also discloses an intermediate compound of formula Vb

wherein, R2 and R3 is selected from carbonyl, amine, alkoxide, Boc protecting group or Fmoc protecting group; R2 and R3 can be bonded together to form a carbocyclic or heterocyclic, saturated or unsaturated ring, which may optionally be mono- or poly-substituted with keto, hydroxy, amino, halogens, radicals containing N, O, P atoms; R2’ and R4 is amine protecting group selected from Fmoc, Boc or Cbz.

In one more aspect present invention discloses an intermediate compound Fragment 2

wherein, R2 and R3 is selected from carbonyl, amine, alkoxide, Boc protecting group or Fmoc protecting group; R2 and R3 can be bonded together to form a carbocyclic or heterocyclic, saturated or unsaturated ring, which may optionally be mono- or poly-substituted with keto, hydroxy, amino, halogens, radicals containing N, O, P atoms; R4 is amine protecting group selected from Fmoc, Boc or Cbz.

EXAMPLES:

The details of the invention are given in the examples provided below, which are given to illustrate the invention only and therefore should not be construed to limit the scope of the invention.

EXAMPLE 1: Process for the preparation of Difelikefalin Acetate by using solution – phase peptide synthesis approach.

Stage (a): Synthesis of Boc-D-Phe-D-Phe-OH (Fragment-1; when R1 is Boc anhydride)

Stage-1a: Synthesis of Boc-D-Phe-OSu
Boc-D-Phe-OH was dissolved in THF and NHS was added and stirred for 10 minutes at 5-10 °C. DIC was dissolved in THF and added to the resulting reaction mixture stirred for 3 hours at 25-30 °C. Precipitated solid was filtered and filtrate was distilled, and IPA was added and precipitated solid was filtered washed with hexane obtain Boc-D-Phe-OSu.

Stage-2a: Synthesis of Boc-D-Phe-D-Phe-OH
D-Phenylalanine was dissolved in base water and stirred for 10 minutes at 5-10 °C. Stage-1a compound was dissolved in THF were added to the resulting reaction mixture at 5-10 °C and stirred for 2 hours at the 25-30 °C. Precipitated solid was filtered and Ethyl Acetate was added, pH of the solution was adjusted to 3 to 4 and organic layer was separated and distilled the solvent and DIPE/MTBE was added precipitated solid was filtered washed with hexane to obtain Boc-D-Phe-D-Phe-OH.

Stage (b): Synthesis of tert-butyl (R)-(5-amino-6-(2,4-dioxo-1,3,8-triazaspiro[4,5] decan-8-yl)-6-oxohexyl)carbamate (Fragment-2; when R2 and R3 together form a cyclo compound and R4 is Boc anhydride)

Stage-1b: Synthesis of benzyl tert-butyl (6-(2,4-dioxo-1,3,8-triazaspiro[4,5]decan-8-yl)-6-oxohexane-1,5-diyl)(R)-dicarbamate
Z-D-Lys(Boc)-OH was dissolved in DMF and stirred for 10 minutes. EDC.HCl and HOAT was added at 5 - 15 °C and stirred for 30 minutes at the same temperature. 1,3,8-triazaspiro[4.5]decane-2,4-dione.HCl was added to the reaction mass and dropwise addition of NMM. The resulting mixture was stirred at 20 to 30 °C. Monitored progress of the reaction by HPLC/TLC. Ethyl Acetate was added and followed by water. Settled the mass for 10 to 15 minutes and organic layer was separated and washed the organic layer with 5% Potassium bisulphate, water, 5% Sodium carbonate and water. Distilled of organic layer under reduced pressure and DIPE/MTBE was and stirred for 2 hours then the white solid was collected by filtration, washed with DIPE, the crude product was further purified on silica gel column and solvent recrystallization and dried in vacuum at 30 °C overnight. Yield: 90%.

Stage-2b: Synthesis of tert-butyl (R)-(5-amino-6-(2,4-dioxo-1,3,8-triazaspiro[4,5] decan-8-yl)-6-oxohexyl)carbamate
Stage-1b compound was dissolved in Methanol and added 5% Pd/C under nitrogen atmosphere. Hydrogen gas was applied and maintained up to completion of reaction. The reaction was monitored by HPLC and filtered the reaction mass under nitrogen atmosphere. Filtrate was distilled off under reduced pressure and DIPE/MTBE was added to obtain a precipitate. Purified the product by solvent crystallization and dried in vacuum at 30 °C overnight.

Stage-3b: Synthesis of tert-butyl ((R)-5-((R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-methylpentanamido)-6-(2,4-dioxo-1,3,8-triazaspiro [4.5]decan-8-yl)-6-oxohexyl)carbamate
Fmoc-D-Leu-OH was dissolved in DMF and stirred for 10 minutes at 25-30 °C. Stage-2b compound, EDC.HCl and HOAT in DMF were added to the resulting reaction mixture at 25-30 °C and stirred for 15-20 minutes at the same temperature. Base water added, precipitated solid was filtered and washed with water and hexane to obtain 2,4-Dioxo,1,3,8-triazaspiro[4,5]decan-8-yl(Fmoc-D-Leu-D-Lys(Boc)).

Stage-4b: Synthesis of tert-butyl ((R)-5-((R)-2-amino-4-methylpentanamido)-6-(2,4-dioxo-1,3,8-triazaspiro[4.5]decan-8-yl)-6-oxohexyl)carbamate
Stage-3b compound was dissolved in Acetonitrile, tert-Butylamine and n-Heptane was added and stirred for 12 hours at 25-30 °C. n-Heptane layer was separated and Acetonitrile layer was distilled and DIPE was added and precipitated solid was filtered and washed with DIPE to obtain 2,4-Dioxo,1,3,8-triazaspiro[4,5]decan-8-yl(NH2-D-Leu-D-Lys(Boc)).

Stage (c): Synthesis of Difelikefalin Acetate

Stage-1c: Synthesis of tert-butyl ((10R,13R,16R,19R)-16-benzyl-10-(2,4-dioxo-1,3,8-triazaspiro[4.5]decane-8-carbonyl)-13-isobutyl-2,2-dimethyl-4,12,15,18-tetraoxo-20-phenyl-3-oxa-5,11,14,17-tetraazaicosan-19-yl)carbamate
Fragment-2 was dissolved in DMF and stirred for 10 minutes at 25-30 °C. Fragment-1, DIC and Oxyma in DMF were added to the resulting reaction mixture at 25-30 °C and stirred for 2-4 hours at the same temperature. Base water added, precipitated solid was filtered and washed with water and hexane to obtain stage-1c compound. Purified the product by solvent crystallization and dried in vacuum at 30 °C overnight.

Stage-2c: Synthesis of NH2-(Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc))Pip-OH
Stage-1c compound was dissolved in Methanol and stirred for 10 minutes at 5-15 °C and LiOH in water was added to the resulting reaction mixture at 25-30 °C and stirred for 2-4 hours at the same temperature. pH was adjusted neutral and product was extracted with suitable solvent and organic layer was washed with brine solution and solvent was distilled and DIPE/MTBE was added, precipitated solid was filtered washed with DIPE to obtain stage-2C compound.

Stage-3c: Synthesis of NH2-(D-Phe-D-Phe-D-Leu-D-Lys)Pip-OH. Salt
Stage-3c(i): Synthesis of NH2-(D-Phe-D-Phe-D-Leu-D-Lys)Pip-OH. TFA
Stage-2c compound was added into TFA containing water and TIPS, the resulting reaction mixture at 25-30 °C and stirred for 2-4 hours at the same temperature and cooled the mass to 5-15 °C and DIPE/MTBE was added. Precipitated solid was filtered and washed with DIPE to obtain stage-3c(i) compound.

Stage-3c(ii): Synthesis of NH2-(D-Phe-D-Phe-D-Leu-D-Lys)Pip-OH. HCl
Stage-2c compound was added into HCl in 1,4-dioxane (or) HCl in isopropyl alcohol, the resulting reaction mixture at ambient temperature and stirred for 2-4 hours at the same temperature and cooled the mass to 5-15 °C and DIPE/MTBE was added. Precipitated solid was filtered and washed with DIPE to obtain stage-3c(ii) compound.

Stage-4c: Synthesis of NH2-(D-Phe-D-Phe-D-Leu-D-Lys)Pip-OH. Acetic acid
Stage-3c compound was purified by preparative HPLC in presence of Acetic acid and followed by Lyophilization to get pure product.

EXAMPLE 2: Process for the preparation of Difelikefalin Acetate by using solution – phase peptide synthesis approach

Stage (a): Synthesis of Fmoc-D-Phe-D-Phe-OH (Fragment-1; when R1 Fmoc)

Stage-1a: Synthesis of Fmoc-D-Phe-OSu
Fmoc-D-Phe-OH was dissolved in THF and NHS was added and stirred for 10 minutes at 5-10 °C. DIC was dissolved in THF and added to the resulting reaction mixture, stirred for 3 hours at 25-30 °C. Precipitated solid was filtered and filtrate was distilled, and IPA was added and precipitated solid was filtered washed with hexane to obtain Fmoc-D-Phe-OSu.

Stage-2a: Synthesis of Fmoc-D-Phe-D-Phe-OH
D-Phenylalanine was dissolved in base water and stirred for 10 minutes at 5-10 °C. Stage-1a compound was dissolved in THF were added to the resulting reaction mixture at 5-10 °C and stirred for 2 hours at the 25-30 °C. Precipitated solid was filtered and Ethyl Acetate was added, pH of the solution was adjusted to 3 to 4 and organic layer was separated and distilled the solvent and DIPE/MTBE was added. Precipitated solid was filtered, washed with hexane to obtain Fmoc-D-Phe-Phe-OH.

Stage (b): Synthesis of tert-butyl (R)-(5-amino-6-(2,4-dioxo-1,3,8-triazaspiro [4,5]decan-8-yl)-6-oxohexyl)carbamate (Fragment-2; when R2 and R3 together form a cyclo compound and R4 is Boc anhydride)

Stage-1b: Synthesis of benzyl tert-butyl (6-(2,4-dioxo-1,3,8-triazaspiro[4.5]decan-8-yl)-6-oxohexane-1,5-diyl)(R)-dicarbamate
Z-D-Lys(Boc)-OH was dissolved in DMF and stirred for 10 minutes. EDC.HCl and HOAT was added at 5-15 °C and stirred for 30 minutes at the same temperature. 1,3,8-triazaspiro[4,5]decane-2,4-dione.HCl was added to the reaction mass and dropwise addition of NMM. The resulting mixture was stirred at 20 to 30° C. Monitored progress of the reaction by HPLC/TLC. Ethyl Acetate was added and followed by water. Settled the mass for 10 to 15 minutes and organic layer was separated and washed the organic layer with 5% Potassium bisulphate, water, 5% Sodium carbonate and water. Distilled of organic layer under reduced pressure and DIPE/MTBE was added and stirred for 2 hours then the white solid was collected by filtration, washed with DIPE, the crude product was further purified on silica gel column and solvent recrystallization and dried in vacuum at 30° C overnight. Yield: 90%.

Stage-2b: Synthesis of tert-butyl (R)-(5-amino-6-(2,4-dioxo-1,3,8-triazaspiro [4,5]decan-8-yl)-6-oxohexyl)carbamate
Stage-1b compound was dissolved in methanol and added 5% Pd/C under nitrogen atmosphere. Hydrogen gas was applied and maintained up to completion of reaction. The reaction was monitored by HPLC and filtered the reaction mass under nitrogen atmosphere. Filtrate was distilled off under reduced pressure and DIPE/MTBE was added to obtain a precipitate. Purified the product by solvent crystallization and dried in vacuum at 30° C overnight.

Stage-3b: Synthesis of tert-butyl ((R)-5-((R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-methylpentanamido)-6-(2,4-dioxo-1,3,8-triazaspiro [4.5]decan-8-yl)-6-oxohexyl)carbamate
Fmoc-D-Leu-OH was dissolved in DMF and stirred for 10 minutes at 25-30 °C. Stage-2b compound, EDC.HCl and HOAT in DMF were added to the resulting reaction mixture at 25-30 °C and stirred for 15-20 minutes at the same temperature. Water was added and precipitated solid was filtered and washed with water and hexane to obtain 2,4-Dioxo,1,3,8-triazaspiro[4,5]decan-8-yl(Fmoc-D-Leu-D-Lys(Boc)).

Stage-4b: Synthesis of tert-butyl ((R)-5-((R)-2-amino-4-methylpentanamido)-6-(2,4-dioxo-1,3,8-triazaspiro[4.5]decan-8-yl)-6-oxohexyl)carbamate
Stage-3b compound was dissolved in Acetonitrile, tert-Butylamine and n-Heptane was added and stirred for 12 hours at 25-30 °C. n-Heptane layer was separated and Acetonitrile layer was distilled and DIPE/MTBE was added and precipitated solid was filtered and washed with DIPE to obtain 2,4-Dioxo,1,3,8-triazaspiro[4,5]decan-8-yl(NH2-D-Leu-D-Lys(Boc)).

Stage (c): Synthesis of Difelikefalin Acetate

Stage-1c: Synthesis of (9H-fluoren-9-yl)methyl ((10R,13R,16R,19R)-16-benzyl-10-(2,4-dioxo-1,3,8-triazaspiro[4.5]decane-8-carbonyl)-13-isobutyl-2,2-dimethyl-4,12, 15,18-tetraoxo-20-phenyl-3-oxa-5,11,14,17-tetraazaicosan-19-yl)carbamate
Fragment-2 was dissolved in DMF and stirred for 10 minutes at 25-30 °C. Fragment-1, EDC.HCl and HOAT in DMF were added to the resulting reaction mixture at 25-30 °C and stirred for 2-4 hours at the same temperature. Base water added, precipitated solid was filtered and washed with water and hexane to obtain stage-1 compound. Purified the product by solvent crystallization and dried in vacuum at 30° C. overnight.

Stage-2c: Synthesis of Boc-(D-Phe-D-Phe-D-Leu-D-Lys)Pip-OH
Stage-1c compound was dissolved in Methanol and stirred for 10 minutes at 25-30 °C. LiOH in water and n-Heptane were added to the resulting reaction mixture at 25-30 °C and stirred for 2-4 hours at the same temperature. pH was adjusted neutral and extracted with dichloromethane. Organic layer was washed with brine solution and solvent was distilled under vacuum.
(or)
Stage-2c(a): Synthesis of Boc-(Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc))Pip-OH
Stage-1c compound was dissolved in IPA and stirred for 10 minutes at 25-30 °C. LiOH in water and n-Heptane was added to the resulting reaction mixture at 25-30 °C and stirred for 2-4 hours at the same temperature. Organic layer was separated, and aqueous layer was washed with DIPE and Boc anhydride was added and to the aqueous layer and the resulting reaction mixture at 25-30 °C and stirred for 2-4 hours. pH was adjusted 3 to 4 and extracted with ethyl acetate. Organic layer was washed with brine and water and solvent was distilled and DIPE/MTBE was added. Precipitated solid was filtered and washed with DIPE to obtain stage-2c compound. Product was crystalized from solvents and dried in vacuum at 30° C overnight.

Stage-3c: Synthesis of NH2-(D-Phe-D-Phe-D-Leu-D-Lys)Pip-OH. Salt
Stage-3c(i): Synthesis of NH2-(D-Phe-D-Phe-D-Leu-D-Lys)Pip-OH.TFA
Stage-2c (or) stage-2c(a) compound was added into TFA containing water and TIPS, the resulting reaction mixture at 25-30 °C and stirred for 2-4 hours at the same temperature and cooled the mass to 5-15° C and DIPE/MTBE was added. Precipitated solid was filtered and washed with DIPE to obtain stage-3c(i) compound.

Stage-3c(ii): Synthesis of NH2-(D-Phe-D-Phe-D-Leu-D-Lys)Pip-OH.HCl
Stage-2c (or) stage-2c(a) compound was added into HCl in 1,4-dioxane (or) HCl in isopropyl alcohol, the resulting reaction mixture at ambient temperature and stirred for 2-4 hours at the same temperature and cooled the mass to 5-15 °C and DIPE/MTBE was added. Precipitated solid was filtered and washed with DIPE to obtain stage-3c(ii) compound.

Stage-4c: Synthesis of NH2-(D-Phe-D-Phe-D-Leu-D-Lys)Pip-OH. Acetic acid
Stage-3c compound was purified by preparative HPLC in presence of Ammonium acetate and followed by Lyophilization to get pure product.

EXAMPLE 3: Process for the preparation of Difelikefalin Acetate by using solution – phase peptide synthesis approach

Stage (a): Synthesis of Boc-D-Phe-D-Phe-OH (Fragment-1; when R1 is Boc anhydride)

Stage-1a: Synthesis of Boc-D-Phe-OSu
Boc-D-Phe-OH was dissolved in THF and NHS was added and stirred for 10 minutes at 5-10 °C. DIC was dissolved in THF and added to the resulting reaction mixture stirred for 3 hours at 25-30 °C. Precipitated solid was filtered and filtrate was distilled, and IPA was added and precipitated solid was filtered washed with hexane obtain Boc-D-Phe-OSu.

Stage-2a: Synthesis of Boc-D-Phe-D-Phe-OH
D-Phenylalanine was dissolved in base water and stirred for 10 minutes at 5-10 °C. Stage-1a compound was dissolved in THF were added to the resulting reaction mixture at 5-10 °C and stirred for 2 hours at the 25-30 °C. Precipitated solid was filtered and Ethyl Acetate was added, pH of the solution was adjusted to 3 to 4 and organic layer was separated and distilled the solvent and DIPE/MTBE was added and precipitated solid was filtered, washed with DIPE to obtain Boc-D-Phe-D-Phe-OH.
Stage (b): Synthesis of Boc-(D-Leu-D-Lys(Boc))Pip-OMe (Fragment-2; when R2 is Boc anhydride, R3 is methoxy group and R4 is Boc anhydride)

Stage 1b: Synthesis of Boc-(Fmoc-D-Lys(Boc))Pip-OMe
Fmoc-D-Lys(Boc)-OH was dissolved in DMF and stirred for 10 minutes. EDC.HCl and HOAT was added at 5 - 15 °C and stirred for 30 minutes at the same temperature. Boc-Pip-OMe was added to the reaction mass and dropwise addition of NMM. The resulting mixture was stirred at 20 to 30° C. Monitored progress of the reaction by HPLC/TLC. Ethyl Acetate was added and followed by water. Settled the mass for 10 to 15 minutes and organic layer was separated and washed the organic layer with 5% potassium bisulphate, water, 5% sodium carbonate and water. Distilled of organic layer under reduced pressure and DIPE/MTBE was and stirred for 2 hours then the white solid was collected by filtration, washed with DIPE, crude product was further purified solvent recrystallization and dried in vacuum at 30° C overnight. Yield: 90%

Stage-2b: Synthesis of Boc-(NH2-D-Lys(Boc))Pip-OMe
Stage-1b compound was dissolved in Acetonitrile, tert-Butylamine and n-Heptane was added and stirred for 12 hours at 25-30 °C. n-Heptane layer was separated, and Acetonitrile layer was distilled, and DIPE/MTBE was added and precipitated solid was filtered and washed with DIPE to obtain Stage-2b compound.

Stage-3b: Synthesis of Boc-(Fmoc-D-Leu-D-Lys(Boc))Pip-OMe
Fmoc-D-Leu-OH was dissolved in DMF and stirred for 10 minutes at 25-30 °C. Stage-2b compound, EDC.HCl and HOAT in DMF were added to the resulting reaction mixture at 25-30 °C and stirred for 15-20 minutes at the same temperature. Water was added and precipitated solid was filtered and washed with water and hexane to obtain stage-3b compound.

Stage-4b: Synthesis of Boc-(NH2-D-Leu-D-Lys(Boc))Pip-OMe
Stage-3b compound was dissolved in Acetonitrile, tert-Butylamine and n-Heptane was added and stirred for 12 hours at 25-30 °C. n-Heptane layer was separated, and Acetonitrile layer was distilled, and DIPE/MTBE was added and precipitated solid was filtered and washed with DIPE to obtain Stage-4b compound.

Stage (c): Synthesis of Difelikefalin Acetate

Stage-1c: Synthesis of Synthesis of tert-butyl ((10R,13R,16R,19R)-16-benzyl-10-(2,4-dioxo-1,3,8-triazaspiro[4.5]decane-8-carbonyl)-13-isobutyl-2,2-dimethyl-4,12,15,18-tetraoxo-20-phenyl-3-oxa-5,11,14,17-tetraazaicosan-19-yl)carbamate.
Fragment-2 was dissolved in DMF and stirred for 10 minutes at 25-30 °C. Fragment-1, EDC.HCl and HOAT in DMF were added to the resulting reaction mixture at 25-30 °C and stirred for 2-4 hours at the same temperature. Water was added and precipitated solid was filtered and washed with water and hexane to obtain stage-1c compound. Purified the product by solvent crystallization and dried in vacuum at 30° C overnight.

Stage-2c: Synthesis of Boc-(Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc))Pip-OH
Stage-1c compound was dissolved in Methanol and stirred for 10 minutes at 5-15 °C and LiOH in water was added to the resulting reaction mixture at 25-30 °C and stirred for 2-4 hours at the same temperature. pH was adjusted to 3 to 4, and product was extracted with Ethyl Acetate solvent and organic layer was washed with brine solution and solvent was distilled and DIPE/MTBE was added, precipitated solid was filtered washed with DIPE to obtain stage-2c compound.

Stage-3c: Synthesis of NH2-(D-Phe-D-Phe-D-Leu-D-Lys)Pip-OH. Salt
Stage-3c(i): Synthesis of NH2-(D-Phe-D-Phe-D-Leu-D-Lys)Pip-OH. TFA
Stage-2c compound was added into Trifluoro acetic acid (TFA) containing water and TIPS, the resulting reaction mixture at 25-30 °C and stirred for 2-4 hours at the same temperature and cooled the mass to 5-15° C and DIPE/MTBE was added. Precipitated solid was filtered and washed with DIPE to obtain stage-3c(i) compound.

Stage-3c(ii): Synthesis of NH2-(D-Phe-D-Phe-D-Leu-D-Lys)Pip-OH. HCl
Stage-2c compound was added into HCl in 1,4-dioxane (or) HCl in isopropyl alcohol, the resulting reaction mixture at ambient temperature and stirred for 2-4 hours at the same temperature and cooled the mass to 5-15° C and DIPE/MTBE was added. Precipitated solid was filtered and washed with DIPE to obtain stage-3c(ii) compound.

Stage-4c: Synthesis of NH2-(D-Phe-D-Phe-D-Leu-D-Lys)Pip-OH. Acetic acid
Stage-3c compound was purified by preparative HPLC in presence of Ammonium acetate and followed by Lyophilization to get pure product.
,CLAIMS:

1. An improved process for the preparation of Difelikefalin Acetate having the structural Formula I, by using solution phase synthesis

which comprises:
i. coupling of Fragment-1 with Fragment-2 in presence of coupling reagent to obtain compound of Formula Ic

wherein R2 and R3 is selected from the group consisting of carbonyl, amine, alkoxide, Boc protecting group or Fmoc protecting group; R2 and R3 can be bonded together to form a carbocyclic or heterocyclic, saturated or unsaturated ring, which may optionally be mono- or poly-substituted with keto, hydroxy, amino, halogens, radicals containing N, O, P atoms; R4 is amine protecting group selected from Fmoc, Boc or Cbz;
ii. deprotection of compound of Formula Ic in presence of a base to obtain compound of Formula IIc;

iii. deprotection of compound of Formula IIc with TFA or HCl to obtain compound of Formula IId as TFA salt or HCl salt; and

iv. purification of compound of Formula IId in presence of Acetic acid or Ammonium acetate to obtain pure Difelikefalin Acetate of compound of Formula I.

2. The process as claimed in claim 1, wherein said coupling reagent is selected from the group consisting of N,N'-Diisopropylcarbodiimide (DIC), Dicyclohexyl carbodiimide (DCC), 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).HCl, 1-Hydroxybenzotriazole (HOBT), 1-Hydroxy-7-azabenzotriazole (HOAT), and mixture thereof.

3. The process as claimed in claim 1, wherein said base is selected from the group consisting of potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, lithium hydroxide, ammonium hydroxide, di-isopropyl amine, N, N-di-isopropyl ethylamine, triethylamine, tertiary butyl amine, piperidine, diethylamine, piperidine in dimethyl formamide and mixture thereof.

4. An improved process for the preparation of compound of Fragment 2, by using solution phase synthesis

wherein, R2 and R3 is selected from carbonyl, amine, alkoxide, Boc protecting group or Fmoc protecting group; R2 and R3 can be bonded together to form a carbocyclic or heterocyclic, saturated or unsaturated ring, which may optionally be mono- or poly-substituted with keto, hydroxy, amino, halogens, radicals containing N, O, P atoms; R4 is amine protecting group selected from Fmoc, Boc or Cbz;

which comprises:
a) coupling of compound of Formula Ib with compound of Formula IIb in presence of coupling reagent to obtain compound of Formula IIIb;

wherein, R2 and R3 is selected from carbonyl, amine, alkoxide, Boc protecting group or Fmoc protecting group or bonded together to form a carbocyclic or heterocyclic, saturated or unsaturated ring, which may optionally be mono- or poly-substituted with keto, hydroxy, amino, atoms containing N, O; R2’ and R4 is amine protecting group selected from Fmoc, Boc or Cbz;
b) deprotection of compound of Formula IIIb to obtain compound of Formula IVb;

c) reacting compound of Formula IVb with amine protected leucine compound to obtain compound of Formula Vb; and

d) deprotection of compound of Formula Vb to obtain compound of Fragment 2.

5. The process as claimed in claim 4, wherein said coupling reagent is selected from the group consisting of N,N'-Diisopropylcarbodiimide (DIC), Dicyclohexyl carbodiimide (DCC), 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).HCl, 1-Hydroxybenzotriazole (HOBT), 1-Hydroxy-7-azabenzotriazole (HOAT), and mixture thereof.

6. A process for the preparation of compound of Formula IIIb

which comprises coupling of compound of Formula Ib with compound of Formula IIb in presence of coupling reagent to obtain compound of Formula IIIb

wherein, R2 and R3 is selected from carbonyl, amine, alkoxide, Boc protecting group or Fmoc protecting group or bonded together to form a carbocyclic or heterocyclic, saturated, or unsaturated ring, which may optionally be mono- or poly-substituted with keto, hydroxy, amino, atoms containing N, O; R2’ and R4 is amine protecting group selected from Fmoc, Boc or Cbz.

7. A process for the preparation of compound of Formula IVb

which comprises deprotection of compound of Formula IIIb to obtain compound of Formula IVb

wherein, R2 and R3 is selected from carbonyl, amine, alkoxide, Boc protecting group or Fmoc protecting group or bonded together to form a carbocyclic or heterocyclic, saturated or unsaturated ring, which may optionally be mono- or poly-substituted with keto, hydroxy, amino, atoms containing N, O; R2’ and R4 is amine protecting group selected from Fmoc, Boc or Cbz.

8. A process for the preparation of compound of Formula Vb

which comprises reaction of compound of Formula IVb with amine protected leucine compound to obtain compound of Formula Vb

wherein, R2 and R3 is selected from carbonyl, amine, alkoxide, Boc protecting group or Fmoc protecting group or bonded together to form a carbocyclic or heterocyclic, saturated or unsaturated ring, which may optionally be mono- or poly-substituted with keto, hydroxy, amino, atoms containing N, O; R2’ and R4 is amine protecting group selected from Fmoc, Boc or Cbz.

9. The process as claimed in claims 1-8, wherein said process employs solvent selected from the group consisting of methanol, ethanol, isopropanol, chloroform, methylene chloride, ethyl acetate, isopropyl acetate, diethyl ether, tetrahydrofuran (THF), 2-methyltetrahydrofuran, diisopropyl ether (DIPE), Methyl tert-butyl ether (MTBE), acetonitrile, dimethylformamide, dimethylacetamide, 1,4-dioxane, N-Methyl-2-pyrrolidone (NMP), acetone, hexane, heptane, water and mixture thereof.

10. An intermediate compound of formula IIIb

wherein, R2 and R3 is selected from carbonyl, amine, alkoxide, Boc protecting group or Fmoc protecting group; R2 and R3 can be bonded together to form a carbocyclic or heterocyclic, saturated or unsaturated ring, which may optionally be mono- or poly-substituted with keto, hydroxy, amino, halogens, radicals containing N, O, P atoms; R2’ and R4 is amine protecting group selected from Fmoc, Boc or Cbz.

11. An intermediate compound of formula IVb

wherein, R2 and R3 is selected from carbonyl, amine, alkoxide, Boc protecting group or Fmoc protecting group; R2 and R3 can be bonded together to form a carbocyclic or heterocyclic, saturated or unsaturated ring, which may optionally be mono- or poly-substituted with keto, hydroxy, amino, halogens, radicals containing N, O, P atoms; R4 is amine protecting group selected from Fmoc, Boc or Cbz.

12. An intermediate compound of formula Vb

wherein, R2 and R3 is selected from carbonyl, amine, alkoxide, Boc protecting group or Fmoc protecting group; R2 and R3 can be bonded together to form a carbocyclic or heterocyclic, saturated or unsaturated ring, which may optionally be mono- or poly-substituted with keto, hydroxy, amino, halogens, radicals containing N, O, P atoms; R2’ and R4 is amine protecting group selected from Fmoc, Boc or Cbz.

13. An intermediate compound Fragment 2

wherein, R2 and R3 is selected from carbonyl, amine, alkoxide, Boc protecting group or Fmoc protecting group; R2 and R3 can be bonded together to form a carbocyclic or heterocyclic, saturated or unsaturated ring, which may optionally be mono- or poly-substituted with keto, hydroxy, amino, halogens, radicals containing N, O, P atoms; R4 is amine protecting group selected from Fmoc, Boc or Cbz.