Abstract: The present invention relates to a method for preparing a peptide Angiotensin-II acetate of formula (1) using solid phase synthesis. It further relates to the solid phase synthesis of Angiotensin-II acetate of formula (1) containing Fmoc and Boc protected amino acids residues.
1. A process for the preparation of Angiotensin-II acetate of formula (1), comprising:
a) Loading first amino acid Fmoc-Phe-OH of formula (18)
on CTC Resin of formula (19) to obtain Fmoc-Phe-CTC Resin of formula
using manual solid phase synthesizer; b) deprotecting Fmoc-Phe-CTC Resin of formula (17) and coupling with the required amino acids to form Fmoc-Asp(0?Bu)-Arg (Pbf)-Val-Tyr(^Bu)-Ileu-Hist(Trt)-Pro-Phe-CTC Resin of formula (3),
in the said order, wherein the order of amino acids may be as Fmoc-L-Pro-OH, Fmoc-His(Trt)-OH, Fmoc-Ileu-OH, Fmoc-Tyr(YBu)-OH, Fmoc-Val-OH, Fmoc-Arg(Pbf)-OH and Fmoc-Asp(OfBu)-OH ; and c) cleaving the protected liner octapeptide Fmoc-Asp (Offiu)-Arg(Pbf)-Val-Tyr(fflu)-Ileu-Hist(Trt)-Pro-Phe-CTC Resin of formula (3) from the CTC resin and deprotecting the side chain protecting groups of intermediate of formula (3) to yield Angiotensin-II trifluoroacetate of formula (2). Finally, trifluoro acetate salt of formula (2) is converted to acetate salt of formula
2. The process as claimed in claim 1, wherein the polymer used in step a) may be selected from a group comprising Chlorotrityl Chloride resin (CTC Resin); Wang Resin also known as p-alkoxy benzyl alcohol resin, 4-(Hydroxymethyl) Phenoxyacetic Acid (HMPA) on Aminoethyl Polystyrene Resins, Rink acid resin, Sasrin linker resin.
3. The amino protecting group is selected from group comprising of Fmoc (9-fluorenyl methoxy carbonyl), Boc (tert-butyloxycarbonyl), Cbz (Benzyloxycarbonyl), Bpoc (2-(4-biphenyl)-2-propyloxycarbonyl), 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf).
4. The process as claimed in claim 1, wherein the hydroxyl protecting group is selected from the group comprising of DMT (dimethoxy trityl), MMT (Methoxytrityl), TRT (Trityl), tert-butyl, t-butoxy carbonyl.
5. The process as claimed in claim 1, wherein the coupling agent (s) is selected from the group comprising of phosgene, carbonyldiimidazole (CDI), HOBt (Hydroxy benzotriazole), TBTU (0-(benzotriazol-l-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate), DCC (1,3-dicyclohexylcarbodiimide), DIC (Diisopropyl carbodiimide), HBTU (O-Benzotriazole-N.N.N'N-tetramethyluronium hexafluoro phosphate), BOP (Benzotriazol-l-yl-oxy-tris(dimethylamino) phosphonium hexafluorophosphate), PyBOP (Benzotriazol-1-yloxy tri(pyrrolidino) phosphonium hexafluorophosphate), PyBrOP (Bromotri (pyrrolidino)phosphonium hexafluorophosphate), Chlorotri(pyrrolidino) phosphonium hexafluorophosphate (PyClOP), Ethyl-2-cyano-2-(hydroxyimino) acetate (OxymaPure), 0-(6-Chlorobenzotriazol-l-yl)-l, 1,3,3-tetramethyluronium tetrafluoroborate (TCTU), Ethyl l,2-dihydro-2-ethoxyquinoline-l-carboxylate (EEDQ), 1 -Cyano-2-ethoxy-2oxoethyHdenaminooxy)dimethylamino morpholino carbenium hexafluorophosphate (COMU), 3-(Diethoxy-phosphoryloxy)-3H-benzo[d][l ,2,3] triazin-4- one (DEPBT), 1-hydroxy 7-azabenzotriazole (HoAt), l-[Bis(dimethylamino)methylene]-lH-l,2,3-triazolo[4,5-b]pyridinium 3-oxidhexafluoro phosphate (HATU), dimethylamino(triazolo[4,5-b]pyridin-3-yloxy)methylidene]-dimethylazanium tetra fluoroborate (TATU) or mixtures thereof.
6. The process as claimed in claim 1, wherein the deprotecting agent used is selected from a group comprising of 4MP (4-methylpiperidine), PP (piperidine), and PZ (piperazine), pyridine, l,8-Diazabicyclo[5.4.0]undec-7-ene (DBU), TFA (trifluro acetic acid), TES (Triethyl silane), TIS (Triisopropyl silane), thioanisole, anisole, EDT(Ethanedithiol), phenol, DMS (Dimethyl sulfide), p-cresol, m-cresol or mixtures thereof.
7. A process for the preparation of Angiotensin-II acetate of formula (1) or its pharmaceutically acceptable salt and intermediate of formula (36) following the steps comprising of:
a) loading first amino acid Boc-Phe-OH of formula (21) on PAM Resin of formula (20) to obtain Boc-Phe- PAM Resin of formula (22) using manual solid phase synthesizer ;
b) deprotection of Boc-Phe-PAM Resin of formula (22) and coupling with the required amino acids to form Boc- L-Asp (OBzl)-L-Arg(Tos)-L-Val-L-Tyr(Bzl)-L-Ileu-L-Hist(DNP)-L-Pro-L-Phe- PAM Resin of formula (36), in the said order, wherein the sequence of amino acids may be as Boc-L-Pro-OH, Boc-His(DNP)OH, Boc-Ileu-OH, Boc-Tyr(Bzl)-OH, Boc-Val-OH, Boc-Arg(Tos)-OH and Boc-L-Asp (OBzl)-OH ; and
c) cleaving the protected liner octapeptide Boc-L-Asp(OBzl)-L-Arg(Tos)-L-Val-L-Tyr(Bzl)-L-Ileu-L-Hist(DNP)-L-Pro-L-Phe-PAM Resin of formula (36), from the PAM resin and deprotection of the side chain protecting groups of intermediate (36) to yield Angiotensin-II hydro fluoride of formula (37).
d) Converting Angiotensin-II hydro fluoride of formula (37) to acetate salt of formula (1)
8. The process according to claim 1 and 7, wherein the Angiotensin (II) trifluoroacetate salt of formula (2) and hydro fluoride salt of formula (37) are converted to acetate salt of formula (1) by treating with acetic acid in preparative HPLC.
9. A process for purification of Angiotensin II acetate of formula (1) with purity greater than 99.0% by HPLC, comprising:
I. mixing Angiotensin II trifluoroacetate salt (2) or Angiotensin-II hydro fluoride of formula (37) in a suitable diluent;
II. eluting the sample through column chromatography;
III. optionally, isolating pure Angiotensin II trifluoroacetate of formula (2) or Angiotensin-II hydro fluoride of formula (37); and
IV. converting Angiotensin II trifluoroacetate of formula (2) or Angiotensin-II hydro fluoride of formula (37) to Angiotensin II acetate of formula (1).
10. The process according to claim 9, wherein Angiotensin (II) acetate obtained is amorphous as characterized by X-Ray diffractogram.
| # | Name | Date |
|---|---|---|
| 1 | 201941018593-PROVISIONAL SPECIFICATION [09-05-2019(online)].pdf | 2019-05-09 |
| 2 | 201941018593-FORM 1 [09-05-2019(online)].pdf | 2019-05-09 |
| 3 | Correspondence by Applicant_Form1_13-05-2019.pdf | 2019-05-13 |
| 4 | 201941018593-FORM 3 [09-05-2020(online)].pdf | 2020-05-09 |
| 5 | 201941018593-ENDORSEMENT BY INVENTORS [09-05-2020(online)].pdf | 2020-05-09 |
| 6 | 201941018593-DRAWING [09-05-2020(online)].pdf | 2020-05-09 |
| 7 | 201941018593-COMPLETE SPECIFICATION [09-05-2020(online)].pdf | 2020-05-09 |
| 8 | 201941018593-Submission Document-21-07-2020.pdf | 2020-07-21 |