DESC:Title of The Invention
Improved process for the preparation of Difelikefalin Acetate.

Field of The Invention
The present invention provides an improved process for the preparation of Difelikefalin Acetate having the chemical Formula I.

The present invention also provides a crystalline Form of Difelikefalin HCl having the chemical Formula II and its process for preparation.

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

Difelikefalin Acetate and its process for the preparation is 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 required to obtain pure Difelikefalin Acetate. This process is highly expensive and commercially not viable.

US 7,842,662 B2, US 8,536,131B2 and CN 108883185 B also discloses the process for the preparation of Difelikefalin Acetate. These processes have several disadvantages with lot of technical difficulties, expensive production costs due to complex purification methods.

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.

Summary of The Invention

The present invention provides an improved process for the preparation of Difelikefalin Acetate.

The present invention provides a cost effective, novel and an efficient process for the preparation of Difelikefalin Acetate by employing linear synthesis in a required sequential condensation of single amino acids on solid support to obtained final peptide with good yields and purity.

The present invention provides an improved process for the preparation of Difelikefalin Acetate compound of Formula I by using solid phase synthesis.

which comprises:
i) anchoring Boc-Pip(Fmoc)-OH to a resin in presence of a base;
ii) selective deprotection of amino acid using a base;
iii) coupling of Fmoc-D-Lys(Boc)-OH to a resin obtained in step-ii) in presence of coupling agent in a solvent to obtain dipeptide resin;
iv) sequential deprotection and coupling of Fmoc-D-Leu-OH, Fmoc-D-Phe-OH, Boc-D-Phe-OH to the obtained resin in step-iii) in presence of a coupling agent and solvent to obtained protected Difelikefalin;
v) cleaving the protected Difelikefalin using a reagent and solvent to obtain crystalline Difelikefalin HCl salt;
vi) purification of crystalline Difelikefalin HCl in presence of ammonium acetate, purified water and acetonitrile and followed by salt exchange with acetic acid to obtain Difelikefalin Acetate.

In another aspect, the present invention relates to crystalline form of Difelikefalin HCl is characterized by:
the presence of peaks in the X-ray powder diffraction diagram at the following angles of refraction 2?: 3.54°, 6.11°, 7.00°, 8.35°, 12.97° and 19.07° the accuracy of the 2? values is in the range of 2?+/-0.2°and it is designated as Form N1; or

the presence of peaks in the X-ray powder diffraction diagram at the following angles of refraction 2?: 3.59°, 6.12°, 6.22°, 7.05°, 8.35°, 18.72°, 19.00°, 20.03°and 22.38° the accuracy of the 2? values is in the range of 2?+/-0.2° and it is designated as Form N2.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure. 1: shows the X-ray powder diffraction diagram of Difelikefalin HCl Crystalline Form N1.
Figure. 2: shows the X-ray powder diffraction diagram of Difelikefalin HCl Crystalline Form N2.
Detailed Description of the Invention

The present invention provides an improved process for the preparation of Difelikefalin Acetate by making appropriate amino acid in required sequence by using solid phase approach with higher yields and purity.

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

The term “peptide” as used herein includes the peptide as well as pharmaceutically acceptable salts of the peptide. Peptide fragments are prepared by using solid phase peptide synthesis through linear approach.

Solid phase peptide synthesis is carried out on an insoluble polymer which is acid sensitive. Acid sensitive resin selected from the group consisting of 2-chloro trityl chloride resin (2-CTC), wang resin, 4-methyltrityl chloride. Preferably using CTC resin. The resin used for the synthesis of Difelikefalin Acetate undergoes swelling in presence of a solvent selected from the group consisting of dichloromethane (MDC), N, N-Dimethylformamide (DMF) and N, N-Diisopropylethylamine (DIPEA) or its mixture.

Solvents used throughout the invention is selected from the group consisting of alcoholic solvents such as methanol, ethanol, isopropanol; chlorinated solvents such as 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 or a 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), Ethyl cyanohydroxyiminoacetate (Oxyma) or mixture thereof.

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, piperidine, N-methyl morpholine (NMM) or 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) or mixture thereof.

According to the present invention, the cleavage of peptide from resin involves treating the protected peptide anchored to a resin with an acid having at least a scavenger. The acid used in the cleavage is hydrochloric acid (HCl). The scavengers used are selected from the group consisting of TIPS, water or mixture thereof.

The protected amino acids are commercially available or may be prepared according to procedures known in the literature.

In one embodiment, the present invention relates to an improved process for the preparation of Difelikefalin Acetate by coupling appropriate amino acids on solid support in a required sequence, deprotection and condensing them in solid phase. Followed by salt formation and purification on reverse phase HPLC, freeze drying and isolation to get pure Difelikefalin Acetate. The schematic description of the process is as shown in Scheme-I.

Scheme I
In step-i), 2-CTC resin was taken in a SPPS reactor and swollen by adding of dichloromethane (MDC), Boc-Pip(Fmoc)-OH, dimethylformamide (DMF) and diisopropylethylamine (DIPEA) was added to the resulting reaction mixture in presence of dichloromethane (MDC) to obtain Boc-Pip(Fmoc)-OCTC Resin of compound of Formula IV.

The reaction temperature may range from 20 °C to 35 °C and preferably at a temperature in the range from 25°C to 30 °C. The duration of the reaction may range from 3 hours to 5 hours, preferably for a period of 4 hours.

In step-ii), deprotecting the Fmoc group in presence of a base, followed by sequential coupling and deprotection of peptide resin with Fmoc-D-Lys(Boc)-OH, Fmoc-D-Leu-OH, Fmoc-D-Phe-OH, Boc-D-Phe-OH in presence of coupling agent and solvent to obtained protected Difelikefalin.

The coupling agent used in this step is DIC, Oxyma pure in dimethylformamide (DMF) and deprotection is carried out using 20% piperidine in dimethylformamide (DMF).

The reaction temperature may range from 20 °C to 35 °C and preferably at a temperature in the range from 25°C to 30 °C. The duration of the reaction may range from 3 hours to 5 hours, preferably for a period of 4 hours.

In step-iii), cleaving the protected Difelikefalin using a reagent and solvent to obtain crude Difelikefalin HCl salt compound of Formula II.

Reagent used in global deprotection is selected from the group consisting of HCl, TIPS, Water, DTT, Thioanisole, EDT, DMS, cresol, phenol, thiocresol, ammonium iodide, 2,2'-(ethylene dioxy)diethane, water or its mixture.

The reaction temperature may range from 0 °C to 5 °C and preferably at a temperature in the range from 5 °C to 10 °C. The duration of the reaction may range from 3 to 7 hours, preferably for a period of 3 to 6 hours.

In step-iv), purification of crude Difelikefalin HCl in presence of ammonium acetate, purified water and acetonitrile and followed by salt exchange with acetic acid to obtain Difelikefalin Acetate.

Another aspect, the present invention relates to crystalline form of Difelikefalin HCl is characterized by:
The presence of peaks in the X-ray powder diffraction diagram at the following angles of refraction 2?: 4.78°, 8.43°, 8.67°, 9.59°, 12.6°, 15.8°, 16.6°, 17.7°, 19.5° and 21.8° the accuracy of the 2? values is in the range of 2?+/-0.2°and it is designated as Form N1; or

The presence of peaks in the X-ray powder diffraction diagram at the following angles of refraction 2?: 4.84°, 4.93°, 8.52°, 8.71°, 9.84°, 16.01°, 16.72°, 17.75°, 19.57°, 22.0° and 23.79° the accuracy of the 2? values is in the range of 2?+/-0.2° and it is designated as Form N2.

While the present invention has been described in terms of its specific embodiments, certain modifications and equivalents will be apparent to those skilled in the art and are intended to be included within the scope of the present invention. The invention is illustrated below with reference to inventive and comparative examples and should not be construed to limit the scope of the invention.

EXPERIMENTAL PORTION

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: Synthesis of Crystalline Difelikefalin Hydrochloride Salt.

Step-A: 2-CTC resin (10 grams) was taken in a SPPS reactor and dichloromethane (50 mL) was added allowed it to swell for 10 minutes.

Step-B: A solution of Boc-Pip(Fmoc)-OH (4.6 grams) and diisopropylethylamine (8.7 mL) in dry dichloromethane (70 mL), dimethylformamide was added to the resin obtained from step-A and stirred for 2 hours at room temperature. After completion of the reaction, the resin was drained and washed with dimethylformamide (60 mL). The resulting resin was deblocked with 20 % piperidine in dimethylformamide (60 mL) for 10 minutes and washed with dimethylformamide (60 mL).

Step-C: Fmoc-D-Lys(Boc)-OH (2.3 grams) was dissolved in dimethylformamide (60 mL) and stirred for 10 minutes. N,N'-diisopropylcarbodiimide (1.5 mL) and 1-hydroxybenzotriazole hydrate (2.0 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step-B and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (60 mL). The resulting resin was deblocked with 20% piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (60 mL).

Step-D: Fmoc-D-Leu-OH (1.7 grams) was dissolved in dimethylformamide (60 mL) and stirred for 10 minutes. N, N'-diisopropylcarbodiimide (1.5 mL) and 1-hydroxybenzotriazole hydrate (2.0 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step-C and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (60 mL). The resulting resin was deblocked with 20% piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (60 mL).

Step-E: Fmoc-D-Phe-OH (1.9 grams) was dissolved in dimethylformamide (60 mL) and stirred for 10 minutes. N, N'-diisopropylcarbodiimide (1.5 mL) and 1-hydroxybenzotriazole hydrate (2.0 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step-C and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests/ chloranil/ TNBS. After completion of the reaction, the resin was drained and washed with dimethylformamide (60 mL). The resulting resin was deblocked with 20% piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (60 mL).

Step-F: Boc-D-Phe-OH (1.3 grams) was dissolved in dimethylformamide (60 mL) and stirred for 10 minutes. N, N'-diisopropylcarbodiimide (1.5 mL) and 1-hydroxybenzotriazole hydrate (2.0 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step-C and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with dimethylformamide (60 mL). The resulting resin was deblocked with 20% piperidine in dimethylformamide (30 mL) for 10 minutes and washed with dimethylformamide (60 mL).

Step-G: Selective cleavage of 2-CTC-resin and global deprotection of Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)-Pip(Boc)-CTC resin in presence of 4M hydrochloride in ethyl acetate (95 mL), tri-isopropyl silane (5 mL) at 5 °C to 10 °C and maintained for 4 to 6 hours. Then peptide precipitated in a mixture of purified water (7.5 mL) and acetone (75 mL) to obtain Crystalline Difelikefalin Hydrochloride.

Example 2: Synthesis of Amorphous Form of Difelikefalin Acetate.

Crystalline Difelikefalin Hydrochloride Salt (5.5 grams) was dissolved in purified water (25 mL) and loaded onto preparative C18 column (50x250 mm, 120 A0). The peptide was purified using a linear gradient of 50 mm ammonium acetate (Buffer A) and acetonitrile: methanol (8:2) (Buffer B) from 5 % to 50% over 100 minutes. The pure fraction containing the Difelikefalin Acetate was pooled. The acetonitrile was evaporated and the aqueous layer was lyophilized to give the Difelikefalin Acetate as white solid. The resulting peptide was analysed by RP-HPLC and confirmed by MALDI or LC-MS.

Advantages of the present invention:

? In present invention HCL is used for global deprotection instead TFA used in prior art. This resulted increase in the purity.
? The present invention reducing the purification time of preparative HPLC when compared to the prior-art process.
? The present invention is economically significant and eco-friendly.
? The present invention reducing the purification and operational cost when compared to the prior-art process.
,CLAIMS:We claim:

1. An improved process for linear solid phase synthesis of Difelikefalin Acetate compound of Formula I by using solid phase synthesis;

which comprises:
i. anchoring Boc-Pip(Fmoc)-OH to a resin in presence of a base;
ii. selective deprotection of amino acid using a base;
iii. coupling of Fmoc-D-Lys(Boc)-OH to a resin obtained in step-ii) in presence of coupling agent in a solvent to obtain dipeptide resin;
iv. sequential deprotection and coupling of Fmoc-D-Leu-OH, Fmoc-D-Phe-OH, Boc-D-Phe-OH to the obtained resin in step-iii) in presence of a coupling agent and solvent to obtained protected Difelikefalin;
v. cleaving the protected Difelikefalin using a reagent and solvent to obtain crystalline form of Difelikefalin HCl salt;
vi. purification of crystalline form of Difelikefalin HCl in presence of ammonium acetate, purified water and acetonitrile and followed by salt exchange with acetic acid to obtain Difelikefalin Acetate.

2. The process as claimed in claim 1, wherein said base 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, ammonium hydroxide and mixture thereof; di isopropyl amine, N,N-di isopropyl ethylamine, tertiary butyl amine, dimethylamine, pyridine, piperidine, N-methyl morpholine (NMM) or mixture thereof.

3. The process as claimed in claim 1, wherein said coupling agent is selected from 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), Ethyl cyanohydroxyiminoacetate (Oxyma) or mixture thereof.

4. The process as claimed in claim 1, wherein said solvent is selected from group consisting of alcoholic solvents such as methanol, ethanol, isopropanol; chlorinated solvents such as methylene chloride; ester solvents such as ethyl acetate; ether solvents such as diethyl ether, tetrahydrofuran (THF), di-isopropyl ether (DIPE), Methyl tert-butyl ether (MTBE); nitrile solvents such as acetonitrile; aprotic solvents and protic solvents such as dimethylformamide, dimethylacetamide, acetone, water or a mixture thereof.

5. A crystalline Form of Difelikefalin Acetate HCl.

6. The crystalline form of Difelikefalin Acetate HCl as claimed in claim 5, is characterized by having a powder X-ray diffractogram comprising reflections at 2-Theta angles of 3.54°, 6.11°, 7.00°, 8.35°, 12.97° and 19.07° ±0.2° 2?, it is designated as Form N1.

7. The crystalline form of Difelikefalin Acetate HCl as claimed in claim 5, is characterized by having a powder X-ray diffractogram comprising reflections at 2-Theta angles of 3.59°, 6.12°, 6.22°, 7.05°, 8.35°, 18.72°, 19.00°, 20.03°and 22.38°±0.2° 2?, it is designated as Form N2.

8. The crystalline form of Difelikefalin Acetate HCl as claimed in claim 5, is prepared by comprising the following steps of
i. anchoring Boc-Pip(Fmoc)-OH to a resin in presence of a base;
ii. selective deprotection of amino acid using a base;
iii. coupling of Fmoc-D-Lys(Boc)-OH to a resin obtained in step-ii) in presence of coupling agent in a solvent to obtain dipeptide resin;
iv. sequential deprotection and coupling of Fmoc-D-Leu-OH, Fmoc-D-Phe-OH, Boc-D-Phe-OH to the obtained resin in step-iii) in presence of a coupling agent and solvent to obtained protected Difelikefalin;
v. cleaving the protected Difelikefalin using a reagent and solvent to obtain crystalline Difelikefalin HCl salt.