AN IMPROVED PROCESS FOR FMOC SYNTHESIS OF ETELCALCETIDE

Abstract

The present invention relates to an improved process for the synthesis of Etelcalcetide and its analogs by solid phase synthesis of Fmoc protected amino acids in a sequential manner, followed by acetylation of terminal D-cys and cleavage of peptide from solid support. The crude heptapeptide thus obtained is reduced using Tris(2-carboxyethyl) phosphine hydrochloride, purified and oxidized with L-cysteine. The oxidized Etelcalcetide is purified and salt exchanged using a one-step reverse phase chromatography process. The purified Etelcalcetide hydrochloride is then precipitated using organic solvents, concentrated and lyophilized to purity of greater than 99.0%.

Claims

1. An improved process for synthesis of Etelcalcetide or salt or precursor thereof as set forth in Formula I by an orthogonal Fmoc strategy comprising of: ##STR00005## i. covalently linking a Fmoc-D-Arg(Pbf)-OH to polystyrene based solid resin support, ii. removing the α-NH2 protecting group from Fmoc-D-Arg(Pbf)-solid support to obtain a free α-NH2 group, iii. coupling the second Fmoc-D-Ala to the D-Arg(Pbf)-solid support, by activating the amino acid by DIC/HOBT in the presence of organic solvent, iv. deprotecting the Fmoc group by a deprotectant, v. repeating steps ii), iii), iv) for assembling the heptapeptide H-D-Cys(Trt)-D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-solid resin support, vi. acetylating the N-terminal group to produce acetylated heptapeptide Ac-D-Cys(Trt)-D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-solid resin support, vii. cleaving the heptapeptide from solid resin support using cleavage cocktail consisting of TFA in the range of 80 to 95% V/V, TIS in the range of 2 to 10% V/V, DMS in the range of 2 to 10% V/V, TCEP in the range of 1 to 5 V/V, water in the range of 1 to 5% V/V, preferably TFA: TIS: DMS: TCEP: water in the ratio of 85: 6.5: 3.5:1.5:1 (%v/v); to obtain crude heptamer-etelcalcetide as set in the formula II of at least 90% peptide purity, ##STR00006## viii. optionally purifying the crude heptamer-etelcalcetide as set in the formula II of step vii) by chromatography wherein said peptide has a purity of ≥98%, ix. oxidizing heptamer-etelcalcetide as set in the formula II of step viii) with free single cysteine in presence of hydrogen peroxide to obtain Etelcalcetide as set forth formula I x. purifying etelcalcetide hydrochloride of step ix) by reverse phase HPLC to a purity of at least 99.8% xi. precipitating, adjusting chloride salt content and lyophilizing the concentrated purified peptide solution of step x).

2. (canceled)

3. The improved process for synthesis of Etelcalcetide or salt or precursor thereof as claimed in claim 1, wherein cleavage of heptapeptide from the solid resin support is carried out using mixture of solvents selected from a group consisting of TFA, TCEP, TIS, Water, DTT, DMS, DODT.

4. The improved process for synthesis of Etelcalcetide or salt or precursor thereof as claimed in claim 1, wherein cleavage of heptapeptide from the solid resin support is done wherein concentration of peptidyl resin is 10-30 ml/g, preferably 20 ml/g.

5. The improved process for synthesis of Etelcalcetide or salt or precursor thereof as claimed in claim 1, wherein the purification of crude heptamer-etelcalcetide to a purity of ≥99% carried by chromatography is by RP-HPLC by isocratic and/or gradient mode.

6. The improved process for synthesis of Etelcalcetide or salt or precursor thereof as claimed in claim 1, wherein the eluent for RP-HPLC purification of crude heptamer-etelcalcetide by gradient mode comprises of perchloric acid buffer system with pH in the range of 3 to 5, preferably 2.5 as an aqueous phase and acetonitrile as an organic phase with isolated yield of at least 50% and purity of at least 99%.

7. The improved process for synthesis of Etelcalcetide or salt or precursor thereof as claimed in claim 1, wherein the purification of etelcalcetide and its conversion to its hydrochloride salt with purity of ≥99.80% by chromatography is by RP-HPLC by isocratic and/or gradient mode.

8. The improved process for synthesis of Etelcalcetide or salt or precursor thereof as claimed in claim 1, wherein the eluent for RP-HPLC purification of Etelcalcetide by gradient mode comprises of Ammonium chloride in Hydrochloric acid buffer system as an aqueous phase with pH in the range of 2 to 5, preferably 3 and an organic phase comprising from solvents selected from methanol, ethanol or acetonitrile preferably methanol with isolated yield of at least 43% and purity of at least 99.8%.

9. The improved process for synthesis of Etelcalcetide or salt or precursor thereof as claimed in claim 1, wherein the precipitation of purified Etelcalcetide hydrochloride is by solvent system comprising of mixture of solvents consisting from group of methanol, ethanol, isopropyl alcohol, acetonitrile, ethyl acetate, acetone either alone or in combination thereof, most preferably combination of ethanol and acetone.

10. The improved process for synthesis of Etelcalcetide or salt or precursor thereof as claimed in claim 1, wherein the precipitation of purified Etelcalcetide hydrochloride is followed by washing the precipitate obtained with an acidified solution of acetone or a mixture of acetone and ethanol, which consists of 0.05% to 2% hydrochloric acid in acetone, or mixture of acetone: ethanol, most preferably 0.25% hydrochloric acid in acetone to ensure the counter ion content of the Etelcalcetide is maintained within a narrow range of 4 to 5 equivalents with respect to the peptide.

11. The improved process for synthesis of Etelcalcetide or salt or precursor thereof as claimed in claim 1, wherein the adjustment of chloride salt content involves addition of chilled solution of dil. Hydrochloric acid in water to the peptide, the concentration of which is maintained at concentration of 50 to 200 mg/ml more preferably 200 mg/ml.

12. The improved process for synthesis of Etelcalcetide or salt or precursor thereof as claimed in claim 1, wherein the lyophilization of the purified Etelcalcetide hydrochloride is done at the high peptide concentration ranging from 50 to 300 mg/ml, more preferably at 200 mg/ml to obtain an amorphous peptide with a high bulk density of 0.6-1.0 g/cm.sup.3.

13. An improved process for synthesis of Etelcalcetide or salt/precursor thereof as set forth in Formula 1 by an orthogonal Fmoc strategy comprising of: i. covalently linking a Fmoc-D-Arg(Pbf)-OH to polystyrene based solid support, ii. removing the α-NH2 protecting group from Fmoc-D-Arg(Pbf)-solid resin support to obtain a free α-NH2 group, iii. coupling the Fmoc-D-Ala to the D-Arg(Pbf)-solid resin support, by activating the amino acid by DIC/HOBT in the presence of organic solvent, iv. deprotecting the Fmoc group by deprotectant, v. repeating steps ii), iii), iv) for assembling the heptapeptide H-D-Cys(Trt)-D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-solid resin support, vi. acetylating the N-terminal group to produce acetylated heptapeptide Ac-D-Cys(Trt)-D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-solid resin support, vii. cleaving the heptapeptide from solid resin support using cleavage cocktail consisting of TFA in the range of 80 to 95% V/V, TIS in the range of 2.5 to 10% V/V, DMS in the range of 2.5 to 10% V/V, preferably TFA: TIS: DMS in the ratio of 90: 6.5: 3.5 (% v/v) to obtain mixture of crude heptamer-etelcalcetide as set in the formula II and dimer-etelcalcetide as set in the formula III, ##STR00007## viii. pretreating the crude mixture of heptamer-etelcalcetide and dimer- etelcalcetide of step vii) with Tris(2-carboxyethyl)phosphine hydrochloride in presence of perchloric acid buffer having concentration ranging from 0.1 to 2% preferably 1% and pH ranging from 2 to 5, preferably 2.5 to obtain heptamer-etelcalcetide of at least 90% peptide purity, ix. purifying the crude heptamer-etelcalcetide as set in the formula II of step viii) by chromatography wherein said peptide has a purity of ≥98%, x. oxidizing heptamer-etelcalcetide as set in the formula II of step ix) with free single cysteine in presence of Hydrogen peroxide to obtain Etelcalcetide as set forth formula I xi. purifying Etelcalcetide hydrochloride of step x) by reverse phase HPLC to a purity of at least 99.8% xii. precipitating, adjusting chloride salt content and lyophilizing the concentrated purified peptide solution of step xi).

14. The improved process for synthesis of Etelcalcetide or salt or precursor thereof as claimed in claim 13, wherein cleavage of heptapeptide from the solid resin support is carried out using mixture of solvents selected from a group consisting of TFA, TCEP, TIS, Water, DTT, DMS, DODT.

15. The improved process for synthesis of Etelcalcetide or salt or precursor thereof as claimed in claim 13, wherein cleavage of heptapeptide from the solid resin support is done wherein concentration of peptidyl resin is10-30 ml/g, preferably 20 ml/g.

16. The improved process for synthesis of Etelcalcetide or salt or precursor thereof as claimed in claim 13, wherein the purification of crude heptamer-etelcalcetide to a purity of ≥99% carried by chromatography is by RP-HPLC by isocratic and/or gradient mode.

17. The improved process for synthesis of Etelcalcetide or salt or precursor thereof as claimed in claim 13, wherein the eluent for RP-HPLC purification of crude heptamer-etelcalcetide by gradient mode comprises of perchloric acid buffer system with pH in the range of 3 to 5, preferably 2.5 as an aqueous phase and acetonitrile as an organic phase with isolated yield of at least 50% and purity of at least 99%.

18. The improved process for synthesis of Etelcalcetide or salt or precursor thereof as claimed in claim 13, wherein the purification of etelcalcetide and its conversion to its hydrochloride salt with purity of ≥99.80% by chromatography is by RP-HPLC by isocratic and/or gradient mode.

19. The improved process for synthesis of Etelcalcetide or salt or precursor thereof as claimed in claim 13, wherein the eluent for RP-HPLC purification of Etelcalcetide by gradient mode comprises of Ammonium chloride in Hydrochloric acid buffer system as an aqueous phase with pH in the range of 2 to 5, preferably 3 and an organic phase comprising from solvents selected from methanol, ethanol or acetonitrile preferably methanol with isolated yield of at least 43% and purity of at least 99.8%.

20. The improved process for synthesis of Etelcalcetide or salt or precursor thereof as claimed in claim 13, wherein the precipitation of purified Etelcalcetide hydrochloride is by solvent system comprising of mixture of solvents consisting from group of methanol, ethanol, isopropyl alcohol, acetonitrile, ethyl acetate, acetone either alone or in combination thereof, most preferably combination of ethanol and acetone.

21. The improved process for synthesis of Etelcalcetide or salt or precursor thereof as claimed in claim 13, wherein the precipitation of purified Etelcalcetide hydrochloride is followed by washing the precipitate obtained with an acidified solution of acetone or a mixture of acetone and ethanol, which consists of 0.05% to 2% hydrochloric acid in acetone, or mixture of acetone: ethanol, most preferably 0.25% hydrochloric acid in acetone to ensure the counter ion content of the Etelcalcetide is maintained within a narrow range of 4 to 5 equivalents with respect to the peptide.

22. The improved process for synthesis of Etelcalcetide or salt or precursor thereof as claimed in claim 13, wherein the adjustment of chloride salt content involves addition of chilled solution of dil. Hydrochloric acid in water to the peptide, the concentration of which is maintained at concentration of 50 to 200 mg/ml more preferably 200 mg/ml.

23. The improved process for synthesis of Etelcalcetide or salt or precursor thereof as claimed in claim 13, wherein the lyophilization of the purified Etelcalcetide hydrochloride is done at the high peptide concentration ranging from 50 to 300 mg/ml, more preferably at 200 mg/ml to obtain an amorphous peptide with a high bulk density of 0.6-1.0 g/cm.sup.3.

Description

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

[0031] FIG. 1 shows analytical RP-HPLC profile on 4.6 mm×250 mm C18, 2.6 μ column of chemically synthesized crude heptamer Etelcalcetide after cleavage by the process of EXAMPLE 2A of the present invention.

[0032] FIG. 2 shows analytical RP-HPLC profile on 4.6 mm×250 mm C18, 2.6 μ column of chemically synthesized crude heptamer Etelcalcetide after cleavage by the process of EXAMPLE 2B of the present invention

[0033] FIG. 3 shows analytical RP-HPLC profile on 4.6 mm×250 mm C18, 2.6 μ column of chemically synthesized crude heptamer Etelcalcetide obtained after reduction by the process of EXAMPLE 3 of the present invention

[0034] FIG. 4 shows analytical RP-HPLC profile on 4.6 mm×250 mm C18, 2.6 μ column of chemically synthesized heptamer Etelcalcetide obtained after purification by the process of EXAMPLE 4 of the present invention

[0035] FIG. 5 shows analytical RP-HPLC profile on 4.6 mm×250 mm C18, 2.6 μ column of chemically synthesized Etelcalcetide obtained after oxidation by the process of EXAMPLE 5 of the present invention

[0036] FIG. 6 shows analytical RP-HPLC profile on 4.6 mm×250 mm C18, 2.6 μ column of chemically synthesized purified Etelcalcetide obtained after purification by the process of EXAMPLE 5 of the present invention

[0037] FIG. 7 shows analytical RP-HPLC profile on 4.6 mm×250 mm C18, 2.6 μ column of chemically synthesized purified Etelcalcetide obtained after purification by the process of EXAMPLE 6 of the present invention

DETAILED DESCRIPTION OF THE INVENTION:

[0038] In a preferred embodiment, the present invention provides an improved process for synthesis of Etelcalcetide or salt or precursor thereof as set forth in Formula I by an orthogonal Fmoc strategy comprising of:

##STR00002## [0039] i. covalently linking a Fmoc-D-Arg(Pbf)-OH to polystyrene based solid resin support, [0040] ii. removing the α-NH2 protecting group from Fmoc-D-Arg(Pbf)-solid support to obtain a free α-NH2 group, [0041] iii. coupling the second Fmoc-D-Ala to the D-Arg(Pbf)-solid support, by activating the amino acid by DIC/HOBT in the presence of organic solvent, [0042] iv. deprotecting the Fmoc group by a deprotectant, [0043] v. repeating steps ii), iii), iv) for assembling the heptapeptide H-D-Cys(Trt)-D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-solid resin support, [0044] vi. acetylating the N-terminal group to produce acetylated heptapeptide Ac-D-Cys(Trt)-D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-solid resin support, [0045] vii. cleaving the heptapeptide from solid resin support using cleavage cocktail consisting of TFA in the range of 80 to 95% V/V, TIS in the range of 2 to 10% V/V, DMS in the range of 2 to 10% V/V, TCEP in the range of 1 to 5% V/V, water in the range of 1 to 5% V/V, preferably TFA: TIS: DMS: TCEP: water in the ratio of 85: 6.5: 3.5:1.5:1 (%v/v); to obtain crude heptamer-etelcalcetide as set in the formula II of at least 90% peptide purity,

##STR00003## [0046] viii. optionally purifying the crude heptamer-etelcalcetide as set in the formula II of step vii) by chromatography wherein said peptide has a purity of ≥98%, [0047] ix. oxidizing heptamer-etelcalcetide as set in the formula II of step viii) with free single cysteine in presence of hydrogen peroxide to obtain Etelcalcetide as set forth formula I [0048] x. purifying Etelcalcetide hydrochloride of step ix) by reverse phase HPLC to a purity of at least 99.8% [0049] xi. precipitating, adjusting chloride salt content and lyophilizing the concentrated purified peptide solution of step x).

[0050] In another preferred embodiment, the present invention provides an improved process for synthesis of Etelcalcetide or salt/precursor thereof as set forth in Formula 1 by an orthogonal Fmoc strategy comprising of: [0051] i. covalently linking a Fmoc-D-Arg(Pbf)-OH to polystyrene based solid support, [0052] ii. removing the α-NH2 protccting group from Fmoc-D-Arg(Pbf)-solid resin support to obtain a free α-NH2 group, [0053] iii. coupling the Fmoc-D-Ala to the D-Arg(Pbf)-solid resin support, by activating the amino acid by DIC/HOBT in the presence of organic solvent, [0054] iv. deprotecting the Fmoc group by deprotectant, [0055] v. repeating steps ii), iii), iv) for assembling the heptapeptide H-D-Cys(Trt)-D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-solid resin support, [0056] vi. acetylating the N-terminal group to produce acetylated heptapeptide Ac-D-Cys(Trt)-D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-solid resin support, [0057] vii. cleaving the heptapeptide from solid resin support using cleavage cocktail consisting of TFA in the range of 80 to 95% V/V, TIS in the range of 2.5 to 10% V/V, DMS in the range of 2.5 to 10% V/V, preferably TFA: TIS: DMS in the ratio of 90: 6.5: 3.5 (%v/v) to obtain mixture of crude heptamer-etelcalcetide as set in the formula II and dimer-etelcalcetide as set in the formula III,

##STR00004## [0058] viii. pretreating the crude mixture of heptamer-etelcalcetide and dimer-etelcalcetide of step vii) with Tris(2-carboxyethyl)phosphine hydrochloride in presence of perchloric acid buffer having concentration ranging from 0.1 to 2% preferably 1% and pH ranging from 2 to 5, preferably 2.5 to obtain heptamer-etelcalcetide of at least 90% peptide purity, [0059] ix. purifying the crude heptamer-etelcalcetide as set in the formula II of step viii) by chromatography wherein said peptide has a purity of ≥98%, [0060] x. oxidizing heptamer-etelcalcetide as set in the formula II of step ix) with free single cysteine in presence of Hydrogen peroxide to obtain Etelcalcetide as set forth formula I [0061] xi. purifying Etelcalcetide hydrochloride of step x) by reverse phase HPLC to a purity of at least 99.8% [0062] xii. precipitating, adjusting chloride salt content and lyophilizing the concentrated purified peptide solution of step xi).

[0063] In preferred embodiment of the invention the process uses TCEP as reducing agent in cleavage cocktail or pre treatment of cleaved peptide with TCEP. Cleavage of heptamer Etelcalcetide from the peptidyl resin leads to the formation of significant levels of dimer Etelcalcetide by the formation of the disulfide bridge between two units of heptamer Etelcalcetide molecules. This reaction can be attributed to the inductive effect of the neighboring amino acids to D-cysteine. The use of TCEP in the cleavage cocktail prevents the formation of the dimer Etelcalcetide and. ence the crude peptide generated is principally composed of only heptamer Etelcalcetide.

[0064] Alternatively the dimer Etelcalcetide in the crude peptide generated without the use of TCEP in the cocktail can be converted completely to heptamer Etelcalcetide by the use of TECP prior to the purification of HPLC. The sample preparation involves the reduction of the dimer Etelcalcetide to heptamer Etelcalcetide and its subsequent purification. As TCEP is a cost effective reducing agent which works in the acidic pH range its use in the cleavage cocktail or in pre purification process is commercially viable and helps generate crude peptide with higher purity.

[0065] Another embodiment of the present invention provides a process wherein the organic solvent used in the coupling is selected from a group consisting of DMF, NMP, mixture of DMF and MDC or any combination thereof, more preferably DMF.

[0066] Still another embodiment of the present invention provides a process wherein cleavage of heptapeptide from the solid resin support is carried out using mixture of solvents selected from a group consisting of TFA, TCEP, TIS, Water, DTT, DMS, DODT.

[0067] Yet another embodiment of the present invention provides a process wherein cleavage of heptapeptide from the solid resin support is done wherein concentration of peptidyl resin is 10-30 ml/g, preferably 20 ml/g.

[0068] In yet another embodiment of the present invention provides a process wherein the purification of crude heptamer-etelcalcetide to a purity of ≥99% carried by chromatography is by RP-HPLC using isocratic and/or gradient mode.

[0069] In another embodiment of the present invention provides the process wherein the eluent for RP-HPLC purification of crude heptamer-etelcalcetide by gradient mode comprises of perchloric acid buffer system with pH in the range of 3 to 5, preferably 2.5 as an aqueous phase and acetonitrile as an organic phase with isolated yield of at least 50% and purity of at least 99%.

[0070] In another embodiment of the present invention provides the process for purification of Etelcalcetide which is a highly hydrophilic peptide owing to the presence of high content of Arginine residues. The hydrophilicity poses challenges to bind the peptide to a reverse phase column for its purification. The use of Perchloric acid buffer system helps to bind such hydrophilic peptides to reverse phase HPLC column which is mediated through the salt formation of perchlorate ion with the guanido nitrogen of Arginine residues in the peptide thus enabling their purification. Further, Perchloric acid is more economically viable at commercial scales as compared to strong ion pairing agents like Alkane Sulphonic acids, which are also difficult to exchange from the peptide during salt exchange step affecting complete counter ion formation.

[0071] In still another embodiment of the present invention provides the process wherein the purification of Etelcalcetide and its conversion to its hydrochloride salt with purity of ≥99.80% by chromatography is by RP-HPLC using isocratic and/or gradient mode.

[0072] In yet another embodiment of the present invention provides the process wherein the eluent for RP-HPLC purification of Etelcalcetide by gradient mode comprises of Ammonium chloride in Hydrochloric acid buffer system as an aqueous phase with pH in the range of 2 to 5, preferably 3 and an organic phase comprising from solvents selected from methanol, ethanol or acetonitrile preferably methanol with isolated yield of at least 43% and purity of at least 99.8%.

[0073] In yet another embodiment of the present invention provides the process wherein the use of Ammonium chloride as a buffer system enables the chloride salt formation of the peptide and also subsequent removal of the perchlorate counter ion from the peptide. The pH of the buffer is maintained at a range wherein aqueous solutions of Etelcalcetide are highly stable. Ammonium chloride buffer is also soluble in ethanol which facilitates its easy removal during the peptide precipitation step.

[0074] Another embodiment of the present invention provides the process wherein the precipitation of purified Etelcalcetide hydrochloride is by solvent system comprising of mixture of solvents consisting from group of methanol, ethanol, isopropyl alcohol, acetonitrile, ethyl acetate, acetone either alone or in combination thereof, most preferably combination of ethanol and acetone.

[0075] Another embodiment of the present invention provides the process wherein the precipitation of Etelcalcetide hydrochloride using a solvent system ensures the removal of excess buffer components from the peptide simultaneously while isolating the peptide. This step offers a more economically and easily scalable process as compared to nanofiltration process involving buffer exchange.

[0076] Another embodiment of the present invention provides the process wherein the predipitation of purified Etelcalcetide hydrochloride is followed by washing the precipitate obtained with an acidified solution of acetone or a mixture of acetone and ethanol, which consists of 0.05% to 2% hydrochloric acid in acetone, or mixture of acetone: ethanol, most preferably 0.25% hydrochloric acid in acetone to ensure the counter ion content of the Etelcalcetide is maintained within a narrow range of 4 to 5 equivalents with respect to the peptide.

[0077] Another embodiment of the present invention provides the process wherein washing of the wet cake of precipitated peptide using acidified acetone allows for the formation of the precise stoichiometric content of the chloride counter ion.

[0078] In yet another embodiment of the present invention provides the process wherein the adjustment of chloride salt content involves addition of chilled solution of dil. HCl in water to the peptide, the concentration of which is maintained at concentration of 50 to 200 mg/ml more preferably 200 mg/ml.

[0079] In yet another embodiment of the present invention provides the process wherein high concentration lyophilization ensures a high bulk density and low surface area of the resulting lyophilised peptide. This helps to prevent significant absorption of moisture, ensuring better long term stability.

[0080] In other embodiment of the present invention provides the process wherein the lyophilization of the purified Etelcalcetide hydrochloride is done at the high peptide concentration ranging from 50 to 300 mg/ml, more preferably at 200 mg/ml to obtain an amorphous peptide with a high bulk density of 0.6-1.0 g /cm.sup.3.

[0081] In other embodiment of the present invention provides the process wherein the chloride content of the peptide significantly impacts the stability of Etelcalcetide resulting in the formation of acid Etelcalcetide or dimer Etelcalcetide impurities. Adjustment of the chloride content prior to lyophilization ensures the precise stoichiometric ratio of the chloride counterion of 4 to 5 with respect to Etelcalcetide is maintained.

[0082] In yet another embodiment of the present invention provides the process of purification of Etelcalcetide hydrochloride with the overall yield of at least 43% and a purity of at least 99.8%.

[0083] Drawings accompanying the specification gives the analytical purity obtained post purification by RP-HPLC at intermittent steps. The foregoing descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various changes and modifications. Any modification, equivalent replacement, Sand improvement made within the spirit and principle of the present invention shall fall within the protection scope of the present invention.

EXAMPLES

[0084] The present invention is described in more detail below with reference to some illustrative examples. It should be understood, however, that these examples serve only to facilitate understanding of the invention but not to restrict the scope of the invention. Unless otherwise specified, the reagents and instruments used in the examples are common commercially available products.

Glossary of terms used in the specification: [0085] AA: Amino acid [0086] CaSR: calcium-sensing receptor [0087] CKD: chronic kidney disease [0088] DBU: 1,8-Diazabicycloundec-7-ene [0089] DCM: dichloromethane [0090] DIC: N, N Di-isopropylcarbodiimide [0091] DIPE: Diisopropyl ether [0092] DIPEA: N,N Di-sisopropyl ethylamine [0093] DMF: Dimethyl formamide [0094] DMS: Dimethyl sulfide [0095] DTT: Dithiothreitol [0096] DODT: 2,2′-(Ethylenedioxy)diethanethiol [0097] EDT: EthaneDithiol [0098] Eq: Equivalent [0099] HBTU: Hydroxybenzotriazole Uronium Salt [0100] HD: Hemodialysis [0101] HoBT: Hydroxybenzotriazole monohydrate [0102] HPLC: High performance liquid chromatography [0103] MBHA: Methylbenzyhydrylamine [0104] MDC: Methylene Dichloride [0105] mmt: Monomethoxytrityl [0106] MTBE: Methyl tertiary butyl ether [0107] NMM: N-methyl morpholine [0108] NMP: N-Methyl pyrrolidone [0109] Npys: 3-nitro-2-pyridinesulfenyl [0110] pbf:2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl [0111] PhSMe: Thioanisole [0112] PTH: Parathyroid hormone [0113] RP-HPLC: Reverse phase high performance liquid chromatography [0114] RT: Room temperature [0115] sHPT: secondary hyperparathyroidism [0116] Spy/S-Pyr: 2-pyridinesulfenyl [0117] StBu: S-tertiary butyl [0118] TCEP: Tris(2-carboxyethyl)phosphine hydrochloride [0119] TIS/TIPS: Tri isopropyl silane [0120] TFA: Trifluoroacetic acid [0121] trt: trityl

[0122] As used herein ther term “D-amino acid” refers to dextro isomer of an amino acid

[0123] As used herein ther term “heptamer Etelcalcetide” refers to 7 D-amino acid sequence as specified in formula II

[0124] As used herein ther term “dimer Etelcalcetide” refers to sequence of 14 D-amino acid as specified in formula III formed due to disulfide bond formation between two heptamer Etelcalcetide

[0125] As used herein ther term “deprotectant” refers to any reagent used for removing the N-α-amino protecting group in the present invention reference is herby made to Fmoc.

[0126] As used herein ther term “orthogonal Fmoc strategy” refers to an approach which uses the base-labile N-Fmoc group for protection of the α-amino function, and acid labile side chain protecting groups.

Chemical Synthesis of Etelcalcetide Hydrochloride Using FMOC Based Solid Phase Peptide Synthesis

EXAMPLE 1: Synthesis of the Heptamer on the Solid Support SPPS Synthesis of Acetyl-D-Cys(Trt)-D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-Rink Amide Resin

[0127] The peptide was synthesized as the peptide amide by the solid phase peptide synthesis technology on the Rink Amide AM resin using Fmoc chemistry.

TABLE-US-00001 Instrument Automated peptide synthesiser Solid Support Fmoc Rink amide AM resin Excess Ratio of Reagents Amino Acid (3.5 Eq):DIC (3.5 Eq): HOBT (3.5 Eq) Coupling solvent DMF Coupling steps Coupling for 60 mins, 6 washes with DMF for 2 minutes each Deprotection solution 20% piperidine in DMF Deprotection step Deprotection for 2 and 10 minutes, 6 washes with DMF for 2 minutes each Capping Solution Acetic Anhydride (5 Eq), NMM (7.5 Eq)

[0128] Fmoc-Rink Amide AM Resin 50.77 g (33 mmol) was swelled in 355 ml DMF for 30 min, drained and deblocked with 355 ml of 20% Piperidine solution in DMF for 2 minutes and 10 minutes respectively. Alternatively, 2% DBU in DMF or Mixture of both 20% Piperidine solution in DMF and 2% DBU in DMF can be used for deblocking. Deblocking of the Fmoc Rink amide AM resin was followed by washings with DMF 350 ml for 6 times. Polystyrene based supports used are selected from group of Rink Amide AM Resin, Rink Amide Resin, Rink Amide MBHA Resin more preferably Rink Amide AM Resin. Loading of Fmoc Rink Amide AM Resin selected was from 0.28-1.12 mmole/g, preferably 0.2 to 0.55 mmol/g, more preferably 0.4 to 0.5 mmole/g.

Step I: Coupling of Fmoc-D-Arg(Pbf)-OH to Rink Amide AM Resin

[0129] 33.1 g of Fmoc-D-Arg(Pbf)-OH (1.5 eq, 5.1 mmol) and 7.6 g of HOBt.H.sub.2O (1.5 eq, 4.96 mmol) was dissolved in 350 ml DMF and transferred in to deblocked Rink amide AM resin. This was followed by dropwise addition of 7.8 ml of DIC (1.5 eq) and stirred for 2 hrs. After coupling, peptidyl resin washed with DMF (350 ml) for 2 min each for 5 cycles. Acetic anhydride (15.9 ml, 5 eq, 16.85 mmol) and NMM (27.1 ml, 7.5 eq, 24.64 mmol) in 350 ml DMF is used for 30 min to carry out capping. The purpose of this step is to cap unreacted amines on rink amide resin so that the next amino acids coupled are not attached to the resin. After capping peptidyl resin is washed with DMF (350 ml) for 2 min each for 5 cycles and again washed with Dichloromethane(350 ml) for 2 min each for 3 cycles and dried under vacuum. Alternative solvent systems used for carrying out this attachment was uronium based reagent in presence of base lke NMM or DIPEA or mixture of NMM and DIPEA in solvents like DMF, MDC or mixtures thereof. [0130] Weight of Fmoc-D-Arg(Pbf)-Rink Amide AM Resin: 61.4 g [0131] Yield: 84%
Step II: Synthesis of Acetyl-D-Cys(Trt)-D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-Rink Amide AM Resin from Fmoc-D-Arg(Pbf)-Rink Amide AM Resin

[0132] The couplings of the remaining amino acids were carried out ,in a similar way, by , repeating the above cycle, till the desired sequence length was attained. The assembly of the peptide chain is carried out in the following manner.

[0133] The Fmoc-D-Arg(Pbf)-Rink Amide AM Resin was weighed on the basis of the scale of the synthesis. 45.41 grams(20 mmol) of Fmoc-D-Arg(Pbf)-Rink Amide AM resin was swollen in DMF. The synthesis of the linear chain on the solid support involves the sequential deblocking of the Fmoc group of the attached amino acid and the activation and coupling of the protected amino acid. The coupling is established using DIC/HOBT in the ratio of AA: DIC: HOBT in 3.5 equivalents each. The reaction was carried out for 1 Hr in organic solvents selected from a group consisting of DMF, NMP, mixture of DMF and MDC or combination of stated solvents, more preferably DMF to improve coupling. Coupling was repeated when Kaiser test showed positive. Unreacted sites on the growing chain were capped using 9.4 ml of acetic anhydride and 16.5 ml of NMM in DMF (5 Eq, 9.97 mmol:5 Eq, 15 mmol).

[0134] The final step involves the deblocking of the Fmoc group of Fmoc-D-Cys (Trt)-OH and acetylation of the amino terminal using 9.4 ml of acetic anhydride and 16.5 ml of NMM as a Base (5 Eq, 9.97 mmol: 7.5 Eq, 15 mmol). The heptamer bound to peptidyl resin is washed with 318 ml of Dichloromethane for 2 min each for 3 cycles and dried under vacuum. [0135] Weight of peptide obtained 68.15 g

EXAMPLE 2: Cleavage of the Acetylated Heptamer of Etelcalcetide from the Peptidyl Resin

[0136] Two different approaches were used for cleaving cleavage cocktails are described below:

A) Cleavage Mixture of TFA: TIS: DMS

[0137] The dried heptamer bound to peptidyl resin obtained from Example 1 was cleaved using a cleavage mixture consisting of TFA: TIS: DMS. Ratio of the solvents used in cocktail ranged for TFA (80 to 95), TIS (2.5 to 10) and DMS (2.5 to 10) (%v/v). Preferred ratio of the solvents used is 90:6.5: 3.5 (%v/v). Concentration of cleavage cocktail ranged from 10-30 ml/g of peptidyl resin; preferably 20 ml/g for 3 to 4 Hrs. 1.36 L of the cocktail is prepared by mixing 1.2 L of TFA, 88.4 ml of TIS and 47.6 ml of DMS. 68.15 g of peptidyl resin is added to the cocktail. Reaction is stirrcd for 4 hours and filtered. The filtrate was concentrated and precipitated in 10 volumes of cold DIPE/MTBE. The precipitate obtained was filtered and washed with DIPE/MTBE and dried under vacuum to obtain the crude peptide. Analytical chromatogram of the crude peptide obtained in this step is shown in the FIG. 1. [0138] Crude Purity: 50% Heptamer Etelcalcetide and 40% Dimer Etelcalcetide [0139] Yield: 18 g [0140] % Yield: 96.7%

B) Cleavage Mixture of TFA: TIS : DMS: TCEP: Water

[0141] The dried peptidyl resin was cleaved using a cleavage mixture comprising of TFA: TIS: DMS: TCEP: Water in the ratio TFA (80 to 95) TIS (2 to 10) DMS (2 to 10) TCEP (1 to 5), Water (0.5 to 5) (%v/v). Preferred ratio of the solvents used is 85: 6.5: 3.5:1.5:1 at a concentration of 10-30 ml/g of peptidyl resin; preferably 20 ml/g for 3 to 4 Hrs.

[0142] 1.36 L of the cocktail was prepared by mixing 1.15 L of TFA, 88.4 ml of TIS and 47.6 ml of DMS., 20.4 g of TCEP and 13.6 ml of Water, 68.15 g of peptidyl resin was added to the precold cocktail. Reaction was stirred for 4 hours and filtered. The filtrate was concentrated and precipitated in 10 volumes of cold DIPE/MTBE. The precipitate obtained was filtered and washed with DIPE/MTBE and dried under vacuum to obtain the crude peptide. Analytical chromatogram of the crude peptide obtained in this step is shown in the FIG. 2. [0143] Crude peptide Purity obtained: 92.5% [0144] Yield: 18.4 g [0145] % Yield: 98.3%

Example 3: Reduction of the Crude Peptide Before Purification

[0146] Crude peptide obtained in Example 2 A, 7 g (7.53 mmol) is dissolved in a 350 ml of aqueous buffer at pH 2.5, preferably 0.1-2% Perchloric acid in water. pH was adjusted to 2.5 with Sodium hydroxide. Concentration of peptide can be used in range from 5 mg/ml to 50 mg/ml more preferably 20 mg/ml.

[0147] 1.3 g (4.52 mmol) of TCEP.HCl is added to the mixture and the pH of the solution was adjusted to 3 to 5.5 preferably 4.5 using TEA/Sodium hydroxide/Ammonia. The reaction is stirred for 35 min to 180 min preferably 75 min either at 10° C. to ambient temperature (RT), pH adjusted to 2.5 to 3.5 using Hydrochloric acid/ Perchloric acid/ Acetic acid/ TFA preferably Perchloric acid.

[0148] Analytical chromatogram of the crude peptide obtained in this step is shown in the FIG. 3. [0149] Purity of Heptamer Etelcalcetide: 93%

Example 4: Purification of Heptamer Etelcalcetide by HPLC

[0150] The crude peptide generated in Example 3 is purified using a Reverse phase HPLC column consisting of C 18, 10 micron 100 A silica. The mobile phase comprised of a buffer of 0.1-3% v/v Perchloric acid pH 2.5 with Sodium hydroxide in water and Acetonitrile. The peptide was purified using a gradient composition of Acetonitrile with the mobile phase A mentioned in the table below. The collected fraction are analyzed by an Analytical HPLC. Analytical chromatogram of the purified peptide obtained in this step is shown in the FIG. 4. [0151] Purity of heptamer Etelcalcetide fraction: 99.8% [0152] Yield: 3.0 g [0153] % Yield: 50%

TABLE-US-00002 Instrument Preparative HPLC Mobile Phase A 1% Perchloric Acid pH 2.5 with Sodium hydroxide HPLC media C18, 10 micron, 100 A Silica Column Dimension 76 mm × 250 mm Flow rate 138 ml/min Detector wavelength 220 nm

Example 5: Oxidation of Heptamer Etelcalcetide to Etelcalcetide and Purification

[0154] 3.85 g of the purified heptamer Etelcalcetide (4.14 mmol) obtained in Example 4 is diluted to obtain a concentration of 0.8-2.0 mg /ml preferably 1.0 mg/ml. Oxidation is carried out by adding 10.1 g (62.15 mmol) of Cysteine hydrochloride monohydrate and adjusting the pH of the mixture to 7 to 8.5 preferably 8.0 using sodium hydroxide solution. 8.0 ml (93.23 mmol) of 35% Hydrogen peroxide is added to the reaction and the reaction is allowed to continue for 15 mins. After the completion of time the reaction pH is acidified to pH 3.0 with perchloric acid and filtered. Analytical chromatogram of the peptide obtained after oxidation reaction in this step is shown in the FIG. 5. The filtered solution is loaded onto HPLC and purified. The HPLC column comprises C 18, 10 micron silica. The mobile phase B comprised of a buffer of 0.05-1.0 M Ammonium Chloride pH 3.0 with HCl. Methanol or Ethanol or Acetonitrile is used as the eluting solvent in the gradient mode to purify the peptide. The collected fractions are analyzed by an Analytical HPLC. Analytical chromatogram of the purified peptide obtained in this step is shown in the FIG. 6. [0155] Purity of Collected fraction: 99.8% [0156] Yield: 3.0 g [0157] % Yield: 43%

TABLE-US-00003 Instrument Semi Preparative HPLC Mobile Phase B 0.1M NH.sub.4Cl pH 3.0 with HCl and Methanol HPLC media C18, 10 micron, 100A Silica Column Dimension 76 mm × 250 mm Flow rate  80 ml/min Detector wavelength 220 nm

Example 6: Isolation and Lyophilization of Etelcalcetide as a Chloride Salt

[0158] Purified Etelcalcetide 3.0 g in eluting buffer obtained from Example 5 was concentrated under reduced pressure and isolated by precipitating it in 1.5 litre alcoholic solvent or with composition of mixture of solvents such as methanol, ethanol, isopropyl alcohol with either acetonitrile, ethyl acetate, acetone. The precipitate is isolated by centrifugation or by filtration. The precipitate is washed with the above solvent mixture and centrifuged to obtain the wet cake. The wet cake obtained is again washed with an acidified solution of acetone or a mixture of acetone and ethanol, which consists of 0.05% to 2% hydrochloric acid in acetone, or mixture of acetone: ethanol, most preferably 0.25% hydrochloric acid in acetone. The precipitate is again centrifuged to obtain a wet cake. The wet cake obtained is dissolved in water and concentrated to 50-300 mg/ml. The concentrated sample is lyophilised to obtain end product as highly purified Etelcalcetide hydrochloride. The lyophilized amorphous product has bulk density of 0.6 to 0.8 g/cm3. Analytical chromatogram of the purified peptide obtained in this step is shown in the FIG. 7. [0159] Purity: 99.8% [0160] Yield: 3.0 g

Example 7: Isolation and Lyophlization of Etelcalcetide as a Chloride Salt

[0161] Purified Etelcalcetide 3.0 g in eluting buffer obtained from Example 5 was concentrated under reduced pressure and isolated by precipitating it in 1.5 liter alcoholic solvent or with composition of mixture of solvents such as methanol, ethanol, isopropyl alcohol with either acetonitrile, ethyl acetate, acetone. The precipitate is isolated by centrifugation or by filtration. The precipitate is washed with the above solvent mixture and centrifuged to obtain the wet cake. The wet cake obtained is dissolved in water and concentrated to 50-300 mg per ml. The concentrated sample at 200mg/ml is mixed with a chilled dilute solution of Hydrochloric acid in water. The Hydrochloric acid is added to water maintained at 2-8° C. at a concentration of 2% to 6% with respect to the peptide. The solution is lyophilised to obtain end product as highly purified Etelcalcetide hydrochloride. The lyophilized amorphous product has bulk density of 0.6 to 0.8 g/cm.sup.3. Analytical chromatogram of the purified peptide obtained in this step is shown in the FIG. 7. [0162] Purity: 99.8% [0163] Yield: 3.0 g