DESC:RELATED APPLICATION This application claims the benefit of priority of our Indian patent application number IN 201621021157, which is incorporated herein by reference. FIELD OF THE INVENTION:
The present invention relates to improved process for the preparation of Icatibant 5 acetate for Formula-I
Formula-I
BACKGROUND OF THE INVENTION: Icatibant is chemically known as D-Arginyl-L-Arginyl-L-prolyl-L [(4R)-4- 10 hydroxyprolyl]-glycyl-L[3-(2-thienyl)alanyl]-L-Seryl-D-(1,2,3,4-tetrahydroisoquinolin-3-ylcarbonyl)-L[(3aS,7aS)-octahydroindol-2-ylcarbonyl]-Larginine or H-D-Arg-Arg-Pro-Hyp-Gly-Thia-Ser-D-Tic-Oic-Arg-OH and having structure of Formula-I
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Formula-I
Icatibant is peptiomimetic drug consisting of ten amino acids, which is a selective and specific antagonist of bradykinin B2 receptors. Icatibant is indicated for the treatment of acute attacks of hereditary 5 angioedema (HAE). US 5648333 discloses Icatibant and its preparation process using solid phase synthesis method. In drug substance synthesis there is always a continuing need for preparation process which can obtain drug substance in high yields, more purity 10 and has low impurities; and which is commercially feasible as well. OBJECTIVE OF INVENTION: An objective of the present invention is to provide a process for preparing Icatibant, which is simple, industrially applicable and robust. Still another objective of the present invention is to provide a process for 15 Preparing Icatibant, which results in high yield and high purity product. SUMMARY OF THE INVENTION: The present invention provides an improved preparation process of Icatibant substantially free of impurities, particularly substantially free of Icatibant (7-10), (des-Pro3) Icatibant, and (D-Pro3) Icatibant. 20
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The present invention provides an isolated Icatibant (7-10), (des-Pro3) Icatibant, (D-Pro3) Icatibant, and (L-Tic7) Icatibant. The present invention relates to Icatibant substantially free of impurities Icatibant (7-10), (des-Pro3) Icatibant, (D-Pro3) Icatibant, and (L-Tic7) Icatibant The present invention provides an improved process of Icatibant with more 5 purity and high yield.
The present invention provides an improved process of Icatibant, wherein amino acids and coupling reagents used are not more than 2 equivalents. Another aspect of the present invention provides a process for the purification of Icatibant applying reversed phase high performance liquid 10 chromatography (RP-HPLC) comprising a first and second chromatography steps with a mixture of an aqueous buffer or aqueous acid with an organic solvent for elution. BRIEF DESCRIPTION OF ABBREVIATIONS: HBTU - 0 -Benzotriazole-N,N,N',N'-tetramethyluroniumhexafluorophosphate 15 Cl-HOBt - 6-chlorol-hydroxy-benzotriazole HOBt -Hydroxy benzotriazole TBTU- 0 -(benzotriazol- 1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate, DCC- 1,3-dicyclohexylcarbodiimide DIC – Diisopropylcarbodiimide 20 HBTU - 0-(benzotriazol-l-yl)-l ,l ,3,3-tetramethyluroniumhexafluorophosphate BOP-Benzotriazol-l-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate PyBOP - Benzotriazol- l-yloxytri(pyrrolidino)phosphonium hexafluorophosphate
PyBrOP - Bromotri(pyrrolidino)phosphonium hexafluorophosphate
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PyClOP - Chlorotri(pyrrolidino)phosphonium hexafluorophosphate (PyClOP), Oxyma - Ethyl-2-cyano-2-(hydroxyimino)acetate (Oxyma Pure), COMU - 1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino mo holinocarbenium hexafluorophosphate
DIPEA - N,N -diisopropylethylamine 5
DMF - N,N -dimethylformamide DCM - Dichloromethane THF - Tetrahydrofuran NMR - N -Methyl pyrrolidine DMA - Dimethylacetamide 10 TFA - Trifluoro acetic acid EDT - Ethanedithiol TIPS/TIS - Triisopropyl silane DTT - Diothreitol DMS - Dimethyl sulfide 15 DMSO - Dimethyl sulfoxide MTBE - Methyltert-butylether MeOH - Methanol IPA - Isopropyl alcohol CTC - Chlorotrityl chloride 20 Fmoc - 9-fluorenylmethoxycarbonyl
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Boc- tert-butoxycarbonyl Cbz – Benzyloxycarbonyl DETAILED DESCRIPTION OF THE INVENTION:
The embodiments of the present invention described below are not intended to be exhaustive or to limit the invention to the precise forms disclosed in the 5 following detailed description. Rather, the embodiments are chosen and described so that others skilled in the art can appreciate and understand the principles and practices of the present invention. The present invention relates to an improved process of preparation of Icatibant using solid phase synthesis comprising the steps of: 10
a) coupling protected amino acid, PG-Arg (Pbf)-OH to a resin to obtain PG-Arg(Pbf)-Resin;
b) removing the protecting group from the amino acid coupled to resin and coupling with PG-Oic-OH to obtain PG-Oic-Arg(Pbf)-Resin;
c) removing the protecting group from the amino acid coupled to resin and 15 coupling with PG-D-Tic-OH to obtain PG-D-Tic-Oic-Arg(Pbf)-Resin;
d) removing the protecting group from the amino acid coupled to resin and coupling with PG-Ser(tBu)-OH to obtain PG-Ser(tBu)-D-Tic-Oic-Arg(Pbf)- Resin;
e) removing the protecting group from the amino acid coupled to resin and 20 coupling with PG-Thi-OH to obtain PG-Thi-Ser(tBu)-D-Tic-Oic-Arg(Pbf)-Resin;
f) removing the protecting group from the amino acid coupled to resin and coupling with PG-Gly-OH to obtain PG-Gly-Thi-Ser (tBu)-D-Tic-Oic-Arg(Pbf)-Resin; 25
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g) removing the protecting group from the amino acid coupled to resin and coupling with PG-Hyp(tBu)-OH to obtain PG-Hyp(tBu)-Gly-Thi-Ser(tBu)-D-Tic-Oic-Arg(Pbf)-Resin;
h) removing the protecting group from the amino acid coupled to resin and coupling with PG-Pro-OH to obtain PG-Pro-Hyp(tBu)-Gly-Thi-5 Ser(tBu)-D-Tic-Oic-Arg(Pbf)-Resin;
i) removing the protecting group from the amino acid coupled to resin and coupling with PG- Arg(Pbf)-OH to obtain PG- Arg(Pbf)-Pro-Hyp(tBu)-Gly-Thi-Ser(tBu)-D-Tic-Oic-Arg(Pbf)-Resin;
j) removing the protecting group from the amino acid coupled to resin and 10 coupling with PG-D-Arg(Pbf)-OH to obtain PG-D-Arg(Pbf)-Arg(Pbf)-Pro-Hyp(tBu)-Gly-Thi-Ser(tBu)-D-Tic-Oic-Arg(Pbf)- resin;
k) removing the protecting group from the amino acid coupled to resin to obtain NH2-D-Arg(Pbf)-Arg(Pbf)-Pro-Hyp(tBu)-Gly-Thi-Ser(tBu)-D-Tic-Oic-Arg(Pbf)- resin; 15
l) cleaving the peptide obtained in step k) from the resin to obtain Icatibant; and
m) purifying the Icatibant .
In an embodiment of the present invention, the PG is a protecting group selected from but not limited to a group selected from Fmoc, Boc, Cbz, and the 20 like. Preferably Fmoc protected solid phase peptide synthesis is used.
In another embodiment of the present invention, the solid phase synthesis is carried out on an resin i.e. insoluble polymer which is acid sensitive. An acid sensitive resin is selected from a group comprising chlorotrityl resin (CTC), Sasrin, Wang Resin, 4-methytrityl chloride, TentaGel S, TentaGel TGA, Rink acid resin, 25 NovaSyn TGT resin,HMPB-AM resin, 4-(2-(amino methyl)-5-methoxy)phenoxy butyric acid anchored to polymeric resin MBHA, 4-(4-(amino methyl)-3-
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methoxy)phenoxy butyric acid anchored to polymeric resin MBHA and 4-(2-(amino methyl)-3,3-dimethoxy)phenoxy butyric acid anchored to polymeric resin MBHA include, most preferred super acid labile resin is 2-chlorotrityl resins.
In yet another embodiment of the present invention, the coupling reagent used in the above process of sequential coupling of amino acid comprises o-(7-5 azabenzotriazol-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate (HATU), o-(benzotriazol-l-0)-l,l,3,3-tetramethyluronium hexafluorophosphate (HBTU), o-(benzotriazol-l-yl)-l,l,3,3-tetramethyluronium tetrafluoroborate (TBTU), benzotriazole-l-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP),benzotriazole-l-yl-oxy-tris-pyrrolidinophosphonium hexafluorophosphate 10 (PyBOP),N,N-bis-(2-oxo-3-oxazolidinyl)phosphonic dichloride (BOP-C1), bromo-trispyrrolidino-phosphonium hexafluorophosphate (PyBroP), 1,3-diisopropyl-carbodiimide (DIC), , PyClOP, Oxyma pure, COMU, HOBt ; said reaction solvent is selected from DMF, DCM, NMP or DMSO in combination between them. The removal of protecting group is carried out by conventional methods, for 15 example the removal of Fmoc protection is carried out using a secondary amine base comprises piperidine, dimethylamine, diethylamine, diphenylamine or mixtures thereof. In still another embodiment of the present invention, removal of protecting groups and polymer support of the peptide may be affected by addition of a strong 20 acidic composition. The acidic composition is preferably based on an acidic material such as TFA, and contains scavenger reagents including, but not limited to, ethanedithiol (EDT), Diothreitol (DTT), TIPS (triisopropylsilane), 2, 2’-(ethylenedioxy)diethane, acetyl cystein, DMS, phenol, cresol and thiocresol or mixture thereof and water. The relative ratio of acidic material to scavenger to 25 water may be from about 90% to about 95% acidic material, from about 2.5% to about 5% scavenger, and from about 2.5% to about 5% water by weight.
In yet another embodiment of the present invention, purification of Icatibant applying reversed phase high performance liquid chromatography (RP-HPLC)
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comprising a first and second chromatography steps with a mixture of an aqueous buffer or aqueous acid with an organic solvent for elution.
In yet another embodiment of the present invention, purification of Icatibant is carried out by successive Reverse Phase HPLC. The RP-HPLC is expediently performed using a commercially available silica gel sorbent as stationary phase. 5 The elution is carried out either by isocratic condition or by gradient mode. Common mobile phases used for elution include, but not limited to, aqueous buffer comprises ammonium acetate buffer, water or water containing acid such as acetic acid (0.1% to 5%), or any miscible combination of water with various organic solvents like THF, acetonitrile and methanol. The purification system preferably 10 employs gradient elution; preferably gradient elution is performed by either increasing or decreasing the amounts of an organic modifier. Suitable organic modifiers include, but are not limited to, acetonitrile, THF, ethanol, methanol, ethanol, n-propanol or iso-propanol. Ultraviolet (UV) absorption of wavelength in the range of 214 nm to 320 nm was used to monitor. 15 In other embodiment of the present invention, the first chromatography step is preferably carried out using ammonium acetate and acetonitrile, second chromatography step using Acetic acid/water; Acetic acid/acetonitrile . In still another embodiment of the present invention, all synthetic steps of the above described process are performed under mild conditions providing 20 products containing a low content of by-products and producing a final product in high yield and high purity. In another aspect the invention relates to an improved process preparation of Icatibant with reduced Icatibant (7-10), (des-Pro3)Icatibant, (D-Pro3)Icatibant and (L-Tic7) Icatibant impurities. 25 In another aspect the invention relates an improved process for preparation of icatibant, wherein the obtained icatibant is substantially free of impurities.
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In an embodiment, icatibant obtained according to the present invention is substantially free of impurities Icatibant (7-10), (des-Pro3)Icatibant, (D-Pro3)Icatibant and (L-Tic7) Icatibant. In another aspect the invention relates to isolated impurities Icatibant (7-10), (des-Pro3)Icatibant, (D-Pro3)Icatibant and (L-Tic7) Icatibant. 5 Structures of the impurities are as shown below: 1. Icatibant (7-10)
Structure: H-Ser-D-Tic-Oic-Arg-OH 2. (des-Pro3)Icatibant Structure: H-D-Arg-Arg-Hyp-Gly-Thi-Ser-D-Tic-Oic-Arg-OH 10 3. (D-Pro3)Icatibant Structure: H-D-Arg-Arg-D-Pro-Hyp-Gly-Thi-Ser-D-Tic-Oic-Arg-OH 4. (L-Tic7) Icatibant Structure: H-D-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-L-Tic-Oic-Arg-OH The word substantially free means that the amount impurities are less than 15 about 0.15% as measured by any known methods such as HPLC. Specifically, the Icatibant prepared according to present invention, as disclosed herein, contains less than about 0.10%, more specifically less than about 0.05%, still more specifically less than about 0.02% of impurity. The successful course of the coupling and de-protection can be monitored 20 by means of the Kaiser Test and Bromophenol Blue test.
Kaiser test is a qualitative test for the presence or absence of free primary amino groups, and it can be a useful indication about the completeness of a coupling step. The test is based on the reaction of ninhydrin with primary amines,
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which gives a characteristic dark blue colour. The test requires minimal amounts of analyte and is completed within a few minutes. In one embodiment the Kaiser Test is performed as below: Kaiser Test Solutions: Reagent A (100 % pyridine): Pour 50 ml of Pyridine into a small reagent bottle and 5 label it “A”.
Reagent B (5 % ninhydrin solution): Dissolve 1.0 g of ninhydrin in 20 mL of Ethanol and pour into a small reagent bottle and label it as “B”.
Reagent C (80% phenol): Dissolve 40 g of phenol in 10 mL of Ethanol and pour it into a small reagent bottle and label it “C”. 10 Kaiser test procedure: Take 10-15 beads of resin in a test tube and wash with ethanol, 2-3 times. To the tube add 2 to 3 drops of Reagent A, 2 to 3 drops of Reagent B, 2 to 3 drops of Reagent C, followed by heating the tubes at 100°C for 5 minutes and then comparing the color with reference to check the completion of coupling reaction. 15 Other test for monitoring coupling is Bromophenol Blue Test. In one embodiment the Bromophenol Blue test is performed as below Bromphenol blue solution: Dissolve 500 mg of bromophenol blue in DMA (dimethyl acetamide) Bromophenol test procedure: Take 10-15 beads of resin in a test tube, wash with 20 ethanol 2-3 times. To the tube add 2 to 3 drops of bromphenol blue solution and then Comparing the color with reference to check the completion of coupling.
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In an embodiment of the present invention, Icatibant preparation is illustrated in the scheme as shown below: The invention will now be described with reference to the following non-limiting examples. 5 EXAMPLES Example I: PROCESS FOR PREPARING ICATIBANT FIRST AMINO ACID ATTACHMENT TO SOLID SUPPORT (RESIN): Step (a): Preparation of Fmoc-Arg (Pbf) -CTC Resin 2-ChloroTrityl Resin (CTC Resin) (5.05 g, 0.98 mmol/g) was transferred to a glass 10 reaction vessel containing a sintered disk. Anhydrous DCM was then added to the glass vessel and drained after 2 min. Next, a clear solution of Fmoc-Arg (Pbf)-OH (1.5 eq) was in dry DCM and added DIPEA (2.0 eq) under ice-water bath, the mixture was left at room temperature for 5 min. Activated amino acid was added to 2-CTC resin and stirred for 3h at room temperature. After completion, reaction 15 mixture was washed with DMF and DCM Then unreacted resin was acylated with MeOH/DCM/DIPEA for 30 min at room temperature. Finally, the resin was washed with DMF (DCM and MeOH to produce Fmoc-Arg(Pbf)-CTC Resins.
AMINO ACID CHAIN ASSEMBLING:
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Step (b): Fmoc-Oic-OH coupling Fmoc-Arg(Pbf)-CTC resin was swelled in DMF for 20 minutes, then added 20% Piperdine in DMF After completion of the Fmoc deprotection, reaction mixture was washed with DMF and DCM Thereafter, resin beads were taken out and checked for Kaiser test (positive) and Bromo phenol blue test (positive). Next, 5 Fmoc-Oic-OH (32.0 eq) and HOBt (2 eq) were taken in 50 ml beaker and dissolved in DMF, then added DIC (3 eq) under ice-water bath , the mixture was left at room temperature for 5 min. Activated amino acid was added to the resin and stirred for 2hrs at room temperature. The resin was washed with DMF, DCM and DMF (thereafter resin beads were taken out and checked for Kaiser Test (negative) and 10 Bromo phenol blue test (negative) to obtain Fmoc-Oic-Arg(Pbf)-CTC Resin. Step (c): Fmoc-D-Tic-OH coupling Fmoc-Oic-Arg(Pbf)-CTC Resin was swelled in DMF for 20 minutes, then added 20% Piperdine in DMF. After completion of the Fmoc deprotection, reaction mixture was washed with DMF and DCM Thereafter, resin beads were taken out 15 and checked for Kaiser Test (positive) and Bromo phenol blue test (positive). Next, Fmoc-D-Tic-OH (2.0 eq) and HOBt (2 eq) were taken in 50 ml beaker and dissolved in DMF, added DIC (3 eq) under ice-water bath , the mixture was left at room temperature for 5 min. Activated amino acid was added to the resin and stirred for 2hrs at room temperature. The resin was washed with DMF, DCM and 20 DMF, thereafter resin beads were taken out and checked for Kaiser Test (negative) and Bromo phenol blue test (negative) to obtain Fmoc-D-Tic-Oic-Arg(Pbf)-CTC Resin. Step (d): Fmoc-Ser (tBu)-OH coupling:
Fmoc-D-Tic-Oic-Arg(Pbf)-CTC Resin was swelled in DMF for 20 minutes, then 25 added 20% Piperdine in DMF. After completion of the Fmoc deprotection, reaction mixture was washed with DMF and DCM Thereafter, resin beads were taken out and checked for Kaiser Test (positive) and Bromo phenol blue test (positive). Next, Fmoc-Ser(tBu)-OH (2.0 eq) and HOBt (2 eq) were taken in 50 ml beaker and
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dissolved in DMF, then added DIC (3 eq) under ice-water bath, the mixture was left at room temperature for 5 min. Activated amino acid was added to the resin and stirred for 4hrs at room temperature. The resin was washed with DMF, DCM and DMF thereafter resin beads were taken out and checked for Kaiser Test (negative) and Bromo phenol blue test (negative) to obtain Fmoc-Ser(tBu)-D-Tic-5 Oic-Arg(Pbf)-CTC Resin. Step (e): Fmoc-Thi-OH coupling: Fmoc-Ser(tBu)-D-Tic-Oic-Arg(Pbf)-CTC Resin was swelled in DMF (20 ml) for 20 minutes, then added 20% Piperdine in DMF. After completion of the Fmoc deprotection, reaction mixture was washed with DMF and DCM. Thereafter, resin 10 beads were taken out and checked for Kaiser Test (positive) and Bromo phenol blue test (positive). Next, Fmoc-Thi-OH (2.0 eq) and HOBt (2 eq) were taken in 50 ml beaker and dissolved in 20 ml DMF, then added DIC (3 eq) under ice-water bath , the mixture was left at room temperature for 5 min. Activated amino acid was added to the resin and stirred for 2hrs at room temperature. The resin was 15 washed with DMF, DCM and DMF, thereafter resin beads were taken out and checked for Kaiser Test (negative) and Bromo phenol blue test (negative) to obtain Fmoc-Thi-Ser(tBu)-D-Tic-Oic-Arg(Pbf)-CTC Resin. Step (f): Fmoc-Gly-OH coupling:
Fmoc-Thi-Ser(tBu)-D-Tic-Oic-Arg(Pbf)-CTC Resin was swelled in DMF for 20 20 minutes, then added 20% Piperdine in DMF. After completion of the Fmoc deprotection, reaction mixture was washed with DMF and DCM. Thereafter, resin beads were taken out and checked for Kaiser Test (positive) and Bromo phenol blue test (positive). Next, Fmoc-Gly-OH (2.0 eq) and HOBt (2 eq) were taken in 50 ml beaker and dissolved in DMF, then added DIC (3 eq) under ice-water bath , the 25 mixture was left at room temperature for 5 min. Activated amino acid was added to the resin and stirred for 2hrs at room temperature. The resin was washed with DMF, DCM and DMF, thereafter resin beads were taken out and checked for
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Kaiser Test (negative) and Bromo phenol blue test (negative) to obtain Fmoc-Gly-Thi-Ser (tBu)-D-Tic-Oic-Arg(Pbf)-CTC Resin. Step (g): Fmoc-Hyp(tBu)-OH coupling: Fmoc-Gly-Thi-Ser(tBu)-D-Tic-Oic-Arg(Pbf)-CTC Resin was swelled in DMF for 20 minutes, then added 20% Piperdine in DMF. After completion of the Fmoc 5 deprotection, reaction mixture was washed with DMF and DCM. Thereafter, resin beads were taken out and checked for Kaiser Test (positive) and Bromo phenol blue test (positive). Next, Fmoc-Hyp(tBu)-OH (2.0 eq) and HOBt (2 eq) were taken in 50 ml beaker and dissolved in DMF,then added DIC (3 eq) under ice-water bath, the mixture was left at room temperature for 5 min. Activated amino acid was 10 added to the resin and stirred for 2hrs at room temperature. The resin was washed with DMF, DCM and DMF, thereafter resin beads were taken out and checked for Kaiser Test (negative) and Bromo phenol blue test (negative) to obtain Fmoc-Hyp(tBu)-Gly-Thi-Ser(tBu)-D-Tic-Oic-Arg(Pbf)-CTC Resin. Step (h): Fmoc-Pro-OH coupling: 15 Fmoc-Hyp(tBu)-Gly-Thi-Ser(tBu)-D-Tic-Oic-Arg(Pbf)-CTC Resin was swelled in DMF for 20 minutes, then added 20% Piperdine in DMF. After completion of the Fmoc deprotection, reaction mixture was washed with DMF and DCM . Thereafter, resin beads were taken out and checked for Kaiser Test (positive) and Bromo phenol blue test (positive). Next, Fmoc-Pro-OH (2.0 eq) and HOBt (2 eq) were 20 dissolved in DMF, then added DIC (3 eq) under ice-water bath, the mixture was left at room temperature for 5 min. Activated amino acid was added to the resin and stirred for 3hrs at room temperature. The resin was washed with DMF, DCM and DMF, thereafter resin beads were taken out and checked for Kaiser Test (negative) and Bromo phenol blue test (negative) to obtain Fmoc-Pro-Hyp(tBu)-25 Gly-Thi-Ser(tBu)-D-Tic-Oic-Arg(Pbf)-CTC Resin. Step (i): Fmoc-Arg(Pbf)-OH coupling:
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Fmoc-Pro-Hyp(tBu)-Gly-Thi-Ser(tBu)-D-Tic-Oic-Arg(Pbf)-CTC Resin was swelled in DMF, then added 20% Piperdine in DMF. After completion of the Fmoc deprotection, reaction mixture was washed with DMF and DCM. Thereafter, resin beads were taken out and checked for Kaiser Test (positive) and Bromo phenol blue test (positive). Next, Fmoc-Arg(Pbf)-OH (2.0 eq) and HOBt (2 eq) were taken 5 in beaker and dissolved in DMF, then added DIC (3 eq) under ice-water bath, the mixture was left at room temperature for 5 min. Activated amino acid was added to the resin and stirred for 2hrs at room temperature. The resin was washed with DMF DCM and DMF thereafter resin beads were taken out and checked for Kaiser Test (negative) and Bromo phenol blue test (negative) to obtain Fmoc-Arg(Pbf)-Pro-10 Hyp(tBu)-Gly-Thi-Ser(tBu)-D-Tic-Oic-Arg(Pbf)-CTC Resin. Step (j): Fmoc-D-Arg(Pbf)-OH coupling: Fmoc-Arg(Pbf)-Pro-Hyp(tBu)-Gly-Thi-Ser(tBu)-D-Tic-Oic-Arg(Pbf)-CTC Resin was swelled in DMF, then added 20% Piperdine in DMF. After completion of the Fmoc deprotection, reaction mixture was washed with DMF and DCM. Thereafter, 15 resin beads were taken out and checked for Kaiser Test (positive) and Bromo phenol blue test (positive). Next, Fmoc-D-Arg(Pbf)-OH (2.0 eq) and HOBt (1.1 g, 2 eq) were dissolved in DMF, then added DIC (3 eq) under ice-water bath, the mixture was left at room temperature for 5 min. Activated amino acid was added to the resin and stirred for 2hrs at room temperature. The resin was washed with DMF 20 DCM and DMF, thereafter resin beads were taken out and checked for Kaiser Test (negative) and Bromo phenol blue test (negative) to obtain Fmoc-D-Arg(Pbf)-Arg(Pbf)-Pro-Hyp(tBu)-Gly-Thi-Ser(tBu)-D-Tic-Oic-Arg(Pbf)-CTC Resin.
Step (k): Fmoc-D-Arg(Pbf)-Arg(Pbf)-Pro-Hyp(tBu)-Gly-Thi-Ser(tBu)-D-Tic-Oic-Arg(Pbf)-CTC Resin was swelled in DMF, then added 20% Piperdine in DMF to 25 the Fmoc-D-Arg(Pbf)-Arg(Pbf)-Pro-Hyp(tBu)-Gly-Thi-Ser(tBu)-D-Tic-Oic-Arg(Pbf)-CTC resin. After completion of the Fmoc deprotection, reaction mixture was washed with DMF, DCM and again DMF. Thereafter, resin beads were taken out and checked for Kaiser Test (positive) and Bromo phenol blue test (positive). Finally, NH2-D-Arg(Pbf)-Arg(Pbf)-Pro-Hyp(tBu)-Gly-Thi-Ser(tBu)-D-Tic-Oic-30
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Arg(Pbf)-CTC resin was washed with Methanol, Diethyl ether and dried it. Obtained Resin was 10.1 gm. Step (l): Cleavage of the peptide from the resin Pour 100 ml cocktail reagent (TFA:TIPS:H2O, 95%:2.5%:2.5%) in 250 ml RBF and placed in ice bath for 15min. Charge NH2-D-Arg(Pbf)-Arg(Pbf)-Pro-5 Hyp(tBu)-Gly-Thi-Ser(tBu)-D-Tic-Oic-Arg(Pbf)-CTC resin (10.1gm) and stirred the reaction at room temperature for 4hrs. After completion of the reaction, the solution was filtered and precipitated by the addition of 10 volumes of diethyl ether (1 L). The obtained product was filtered and washed with diethyl ether and dried under vacuum to obtain crude peptide (4.6 g). 10 Step (m): Purification and Lyophilization Step (m(i)): The crude peptide solution was passed through Waters RP-HPLC system, wavelength 220 nm, 250 X 50 mm column of CI8 reverse phase column, mobile phase: A : 0.01 M ammonium acetate ; mobile phase B: acetonitrile flow rate: 50 15 ml / min, peaks of fractions were collected and pooled the fractions having the purity greater than 98% and Lyophilized it. Yield: 1.2 g. HPLC purity: 98.0 %. The product was lyophilized to obtain the white powder. Step (m(ii)): The obtained lyophilized product was again passed through Waters RP-HPLC 20 system, wavelength 220 nm, 250 X 50 mm column of C18 reverse phase column, mobile phase: A: 1% acetic acid in water; mobile phase B: acetonitrile flow rate: 50 ml / min, fractions collected and analyzed by HPLC and Qda the fractions having the purity greater than 99% were collected, pooled together, concentrated using rotary evaporation and lyophilized to obtain pure peptide (Icatibant acetate). 25 Example II:
PROCESS FOR PREPARING ICATIBANT
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FIRST AMINO ACID ATTACHMENT TO SOLID SUPPORT (RESIN): Step (1): Preparation of Fmoc-Arg (Pbf) -CTC Resin 2-ChloroTrityl Resin (CTC Resin) (100.0 g, 0.98 mmol/g) was transferred to a glass reaction vessel containing a sintered disk. Anhydrous DCM was then added to the glass vessel and drained after 30 min. Next, a clear solution of Fmoc-Arg 5 (Pbf)-OH (1.2 eq) was taken in a 1Lt beaker and dissolved in dry DCM and added DIPEA (3.0eq) under ice-water bath, the mixture was left at room temperature for 15 min. Activated amino acid was added to 2-CTC resin and stirred for 2h at room temperature. After completion, reaction mixture was washed with DCM. Then unreacted resin was acylated with MeOH/DCM/DIPEA for 30 min at room 10 temperature. Finally, the resin was washed with DMF, DCM and DMF to produce Fmoc-Arg(Pbf)-CTC Resins. AMINO ACID CHAIN ASSEMBLING: Step (2): Fmoc-Oic-OH coupling Fmoc-Arg(Pbf)-CTC resin was swelled in DMF for 5 minutes, then added 20% 15 Piperdine in DMF. After completion of the Fmoc deprotection, reaction mixture was washed with DMF. Thereafter, resin beads were taken out and checked for Kaiser test (positive) and Bromo phenol blue test (positive). Next, Fmoc-Oic-OH (2.0 eq) and HOBt (2 eq) were taken in 1Lt beaker and dissolved in 800 ml DMF, then added DIC (3 eq) under ice-water bath, the mixture was left at room 20 temperature for 15 min. Activated amino acid was added to the resin and stirred for overnight at room temperature. The resin was washed with DMF (3 X 800 ml), DCM and DMF thereafter resin beads were taken out and checked for Kaiser Test (negative) and Bromo phenol blue test (negative) to obtain Fmoc-Oic-Arg(Pbf)-CTC Resin. 25
Remaining aminoacids (Fmoc-D-Tic-OH; Fmoc-Ser(tBu)-OH; Fmoc-Thi-OH; Fmoc-Gly-OH; Fmoc-Hyp(tBu)-OH;Fmoc-Pro-OH; Fmoc-Arg (Pbf)-OH and Fmoc-D-Arg (Pbf)-OH) were sequentially coupled in similar manner to get desired
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NH2-D-Arg(Pbf)-Arg(Pbf)-Pro-Hyp(tBu)-Gly-Thi-Ser(tBu)-D-Tic-Oic-Arg(Pbf)-CTC resin Obtained Resin was 180.0 gm. Step (3): Cleavage of the peptide from the resin Pour 1800 ml cocktail reagent (TFA:TIPS:H2O, 95%:2.5%:2.5%) in 5.0Lt RBF and placed in ice bath for 15min. Charge NH2-D-Arg(Pbf)-Arg(Pbf)-Pro-5 Hyp(tBu)-Gly-Thi-Ser(tBu)-D-Tic-Oic-Arg(Pbf)-CTC resin (180.0gm) and stirred the reaction at room temperature for 3 h. After completion of the reaction, the solution was filtered and precipitated by the addition of 10 volumes of diethyl ether. The obtained product was filtered and washed with diethyl ether and dried under vacuum to obtain crude peptide (66 g). 10 Step (4): Purification and Lyophilization (MORE FEASIBLE APPROACH) Acetate conversion (Ion-Exchange): The crude peptide solution was passed through RP-HPLC system (wavelength 220 nm, 250 X 50 mm column of C18 reverse phase column, mobile phase A: 10 mM ammonium acetate ; mobile phase B: Water, flow rate: 80 ml / min). A gradient 15 method was followed (mobile phase A for 20 min and mobile phase B for 10 min). Gradient Elution: Ion-exchanged peptide was eluted by gradient method (mobile phase A: 0.1% acetic acid in water; mobile phase B: 40% mobile phase A and 60% of acetonitrile, flow rate: 100 ml / min). Fractions collected and analyzed by HPLC and Qda. The 20 fractions having purity greater than 99% were collected, pooled together, concentrated using rotary evaporation and lyophilized to obtain pure peptide (Icatibant acetate 21.0gm). ,CLAIMS:A process for preparing Icatibant substantially free of Icatibant (7-10), (des-Pro3)Icatibant, (D-Pro3)Icatibant and (L-Tic7) Icatibant using solid phase synthesis comprising the steps of :
a) coupling protected amino acid to a resin to obtain amino acid coupled to 5 resin PG-Arg(Pbf)-Resin;
b) removing the protecting group from the amino acid coupled to resin and coupling with PG-Oic-OH to obtain PG-Oic-Arg(Pbf)-Resin;
c) removing the protecting group from the amino acid coupled to resin and coupling with PG-D-Tic-OH to obtain PG-D-Tic-Oic-Arg(Pbf)-Resin; 10
d) removing the protecting group from the amino acid coupled to resin and coupling with PG-Ser(tBu)-OH to obtain PG-Ser(tBu)-D-Tic-Oic-Arg(Pbf)- Resin;
e) removing the protecting group from the amino acid coupled to resin and coupling with PG-Thi-OH to obtain PG-Thi-Ser(tBu)-D-Tic-Oic-15 Arg(Pbf)-Resin;
f) removing the protecting group from the amino acid coupled to resin and coupling with PG-Gly-OH to obtain PG-Gly-Thi-Ser (tBu)-D-Tic-Oic-Arg(Pbf)-Resin;
g) removing the protecting group from the amino acid coupled to resin and 20 coupling with PG-Hyp(tBu)-OH to obtain PG-Hyp(tBu)-Gly-Thi-Ser(tBu)-D-Tic-Oic-Arg(Pbf)-Resin;
h) removing the protecting group from the amino acid coupled to resin and coupling with PG-Pro-OH to obtain PG-Pro-Hyp(tBu)-Gly-Thi-Ser(tBu)-D-Tic-Oic-Arg(Pbf)-Resin; 25
i) removing the protecting group from the amino acid coupled to resin and coupling with PG- Arg(Pbf)-OH to obtain PG- Arg(Pbf)-Pro-Hyp(tBu)-Gly-Thi-Ser(tBu)-D-Tic-Oic-Arg(Pbf)-Resin;
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j) removing the protecting group from the amino acid coupled to resin and coupling with PG-D-Arg(Pbf)-OH to obtain PG-D-Arg(Pbf)-Arg(Pbf)-Pro-Hyp(tBu)-Gly-Thi-Ser(tBu)-D-Tic-Oic-Arg(Pbf)- resin;
k) removing the protecting group from the amino acid coupled to resin to obtain NH2-D-Arg(Pbf)-Arg(Pbf)-Pro-Hyp(tBu)-Gly-Thi-Ser(tBu)-D-5 Tic-Oic-Arg(Pbf)- resin; and
l) cleaving the peptide obtained in step k) from the resin to obtain Icatibant and optionally converting to its acetate salt.
2. The process according to claim 1, wherein the resin is selected from group consisting of chlorotrityl resin (CTC), Sasrin, Wang Resin, 4-methytrityl 10 chloride, TentaGel S, TentaGel TGA, Rink acid resin, NovaSyn TGT resin,HMPB-AM resin, 4-(2-(amino methyl)-5-methoxy)phenoxy butyric acid anchored to polymeric resin MBHA, 4-(4-(amino methyl)-3-methoxy)phenoxy butyric acid anchored to polymeric resin MBHA and 4-(2-(amino methyl)-3,3-dimethoxy)phenoxy butyric acid anchored to 15 polymeric resin MBHA.
3. The process according to claim 1, wherein the resin is chlorotrityl (CTC) resin.
4. The process according to claim 1, wherein PG is the protecting group selected from is Fmoc, Boc, or Cbz. 20
5. The process according to claim 1, wherein the coupling is carried out in presence of coupling agent selected from the group consisting of o-(7-azabenzotriazol-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate (HATU), o-(benzotriazol-l-0)-l,l,3,3-tetramethyluronium hexafluorophosphate (HBTU), o-(benzotriazol-l-yl)-l,l,3,3-25 tetramethyluronium tetrafluoroborate (TBTU), benzotriazole-l-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP),benzotriazole-l-yl-oxy-tris-pyrrolidinophosphonium hexafluorophosphate (PyBOP),N,N-bis-(2-oxo-3-oxazolidinyl)phosphonic
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dichloride (BOP-C1), bromo-trispyrrolidino-phosphonium hexafluorophosphate (PyBroP), 1,3-diisopropyl-carbodiimide (DIC), , PyClOP, Oxyma pure, COMU, HOBt.
6. The process according to claim 1, wherein the steps of removing of protecting groups is carried out using a secondary amine base comprising 5 piperidine, dimethylamine, diethylamine, diphenylamine or mixtures thereof.
7. The process according to claim 1, the cleavage step l) is performed by addition of a strong acidic composition comprising TFA, scavenger reagents selected from ethanedithiol (EDT), Diothreitol (DTT), TIPS 10 (triisopropylsilane), 2, 2’-(ethylenedioxy)diethane, acetyl cystein, DMS, phenol, cresol and thiocresol or mixture thereof and water.
8. The process according to claim 1, the cleavage step l) is performed using a composition of TFA:TIPS:H2O (95%:2.5%:2.5%).
9. The process according to claim 1 further comprising purifying the crude 15 icatibant obtained in step l) through RP-HPLC using mobile phase A: ammonium acetate and mobile phase B: water to obtain Ion-exchanged peptide; followed by gradient elution using mobile phase A: 0.1% acetic acid in water, mobile phase B: 40% mobile phase A and 60% of acetonitrile; and lyophilized to obtain pure Icatibant acetate