WO2020208650A1 - A process for preparing abaloparatide - Google Patents

A process for preparing abaloparatide Download PDF

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Publication number
WO2020208650A1
WO2020208650A1 PCT/IN2020/050338 IN2020050338W WO2020208650A1 WO 2020208650 A1 WO2020208650 A1 WO 2020208650A1 IN 2020050338 W IN2020050338 W IN 2020050338W WO 2020208650 A1 WO2020208650 A1 WO 2020208650A1
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Prior art keywords
leu
fmoc
resin
dmf
tbu
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PCT/IN2020/050338
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French (fr)
Inventor
Jorge BACARDIT CABADO
Gaurav TANEJA
Vijay VISHWAKARMA
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Hemmo Pharmaceuticals Pvt. Ltd.
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Publication of WO2020208650A1 publication Critical patent/WO2020208650A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/635—Parathyroid hormone (parathormone); Parathyroid hormone-related peptides

Definitions

  • the present invention relates to an improved process for the preparation of Abaloparatide having Seq Id No.1.
  • Osteoporosis in postmenopausal women represents a disease burden for which diagnosis and treatment should be healthcare priorities.
  • An osteoporotic fracture can be a life-altering event for a woman and her family.
  • Abaloparatide is an analog of parathyroid hormone related protein ( PTHrP ) and is a synthetic 34 amino acid peptide. It was approved in 2017 by the FDA (marketed under the trade name Tymlos) for the treatment of postmenopausal women with osteoporosis at a high risk for fracture.
  • Abaloparatide is a synthetic peptide that is related to human PTHrP ( hPTHrP ) and its potential has been demonstrated in preclinical testing to widen the anabolic window for bone therapeutics, stimulating bone formation with a limited effect on bone resorption and mineral mobilization. This could enable improved convenience over currently available anabolic therapies, resulting in greater compliance and, ultimately, greater benefit to patients.
  • SPPS Solid phase peptide synthesis
  • CN 106146648 discloses a process to prepare parathyroid hormone analogue (Abaloparatide) by synthesizing separately three fragments (1-15) and (16-23) and (24-33) and then coupling the said fragments to obtain the Abaloparatide.
  • This Chinese patent application makes use of three separately synthesized fragments on three different resins which increase the cost of the entire process.
  • the use of different resins in the synthesis of Abaloparatide fragments causes the process to be tedious and inconvenient. Additionally, the process disclosed therein is prone to isomerization leading to optically impure Abaloparatide which is difficult to purify due to its homogeneity.
  • the present invention provides a process for the synthesis of Abaloparatide represented by Seq Id No.1 comprising;
  • an improved process for preparing Abaloparatide wherein the disadvantage of peptide aggregation on to the solid support as observed in the conventional processes of solid phase synthesis for long chain peptides is overcome by involving dipeptide units.
  • Abaloparatide prepared according to the process of the present invention having purity of 80% to 85% prior to the purification step and >99% purity after the purification step.
  • a pharmaceutical composition comprising Abaloparatide prepared according to the process of the present invention together with one or more pharmaceutically acceptable excipients.
  • FIG. 1 depicts the scheme for the solid phase peptide synthesis (SPPS) for Abaloparatide
  • Figure 2 depicts the HPLC chromatogram of Crude Abaloparatide obtained by the process of the present invention
  • Figure 3 depicts the HPLC chromatogram for % purity of Abaloparatide synthesized on use of rink amide resin with the standard substitution value of 0.50-0.60 mmol/gm;
  • Figure 4 depicts the HPLC chromatogram of purified Abaloparatide obtained by the present invention
  • Figure 5 depicts the mass of purified Abaloparatide thereby confirming the formation of Abaloparatide
  • Figure 6 depicts the purity of crude Abaloparatide obtained by in the absence of Fmoc-Lys(Boc) Ser[Psi(Me,Me)Pro]-OH unit.
  • DIPEA Diisopropylethylamine
  • NMM N-methyl morpholine
  • RT room or ambient temperature
  • TIPS triisopropylsilane
  • Trp tryptophan
  • Trt trityl or triphenylmethyl
  • the present invention relates to a process for preparing Abaloparatide by elongation of Fmoc protected fragments, simultaneous cleavage and de-protection of the peptide chain, purification and isolation of Abaloparatide.
  • the present invention provides a process for the synthesis of Abaloparatide represented by Seq Id No.1 comprising;
  • SPPS solid-phase peptide synthesis
  • the protecting group is selected from the group consisting of traditional Fmoc/tBu protection or Boc/benzyl protection.
  • Other protecting groups such as Cbz, Bpoc could also be used as amino protecting group.
  • the protecting group for the amino acid/dipeptide unit is Fmoc.
  • the present invention provides Fmoc protected amino acid selected from the group comprising Fmoc-Ala-OH; Fmoc-Thr(tbu)- OH; Fmoc-His(Trt)-OH; Fmoc-Leu-OH; Fmoc-Lys(Boc)-OH, Fmoc-Leu-Aib- OH optionally for position (28) and (29); Fmoc-Glu(tbu)-OH; Fmoc-Arg(Pbf)- OH; Fmoc-Asp(OtBu)-OH; Fmoc-Gln-OH; Fmoc-Ile-OH; Fmoc-Lys(boc)- Ser[Psi(Me,Me)Pro]-OH optionally for position (13) and (14); Fmoc-Gly-OH; Fmoc-Ser(
  • the present invention provides Fmoc-Lys(boc)- Ser[Psi(Me,Me)Pro]-OH is a Dimethyloxazolidine dipeptide.
  • Dimethyloxazolidine dipeptides introduce into the peptide chain a pseudoproline residue, derived from either Ser or Thr, which disrupts peptide chain aggregation in the same manner as proline.
  • the present invention used Fmoc-Leu-Aib-OH dipeptide unit to avoid the possible deletion impurities which can be formed due to incomplete coupling of Fmoc-Leu28-OH after Fmoc-Aib29-OH Coupling to the peptide chain bound to the rink amide resin in SPPS.
  • Fmoc-Leu-Aib-OH used in the present process is to overcome the difficulty observed in the detection of Fmoc removal of Fmoc-Aib29- from the peptide chain bound to the resin in the synthesis of Abaloparatide.
  • Fmoc-Aib29-OH After coupling of Fmoc-Aib29-OH on the growing chain of Abaloparatide during the Solid phase, it has been observed that after the deprotection of N-Fmoc group of Fmoc-Aib29- OH by 20% Piperidine/DMF mixture, Kaiser test was not positive i.e. resin beads were not showing expected blue colour instead they showed yellow colour with clear solution (-ive test).
  • the solid support is a resin, wherein the said resin is preferably a rink amide resin.
  • the said rink amide resin is selected from the group comprising 4-methylbenzhydrylamine resin (MBHA), Rink amide BHA resin or Sieber resin., preferably Rink amide MBHA resin.
  • the 4-methylbenzhydrylamine resin (MBHA) is the most preferable resin being used having a low substitution value of 0.1 to 0.4 mmol/g.
  • the coupling agents in the process is selected from the group comprising hydroxybenzotriazole (HOBt), N, N'- diisopropylcarbodiimide (DIC), O-(Benzotriazol-1-yl)-N,N,N',N'- tetramethyluronium tetrafluoroborate (TBTU), N,N,N ⁇ ,N ⁇ -Tetramethyl-O-(1H- benzotriazol-1-yl)uronium hexafluorophosphate (HBTU), 1,3- dicyclohexylcarbodlimide (DCC), 1-(dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC HCl), benzotriazol-1-yl-oxy-tris(dimethyl-amino)- phosphonium hexafluorophosphate (BOP), N,N-bis-(2-oxo-3- oxazolidin
  • the coupling takes place in presence of a solvent selected from the group comprising of DMF, DCM, THF, NMP, DMAC methanol, ethanol, isopropanol, dichloroethane, 1 ,4- dioxane, 2-methyl tetrahydrofuran ethyl acetate, acetonitrile, acetone, and the like or a mixtures thereof.
  • a solvent selected from the group comprising of DMF, DCM, THF, NMP, DMAC methanol, ethanol, isopropanol, dichloroethane, 1 ,4- dioxane, 2-methyl tetrahydrofuran ethyl acetate, acetonitrile, acetone, and the like or a mixtures thereof.
  • Fmoc is removed from the peptide in the presence of an organic base prepared in an organic solvent.
  • the organic base is selected from the group comprising piperidine, piperazine, N-methyl morpholine, diethyl amine, triethyl amine, 1,8-Diazabicyclo [5.4.0]undec-7-ene (DBU) and the like or a mixtures thereof.
  • the organic solvent is selected from the group comprising dimethyl formamide (DMF), N-Methyl-2-Pyrrolidone (NMP), dichloromethane (DCM), tetrahydrofuran (THF), N,N-dimethylacetamide (DMAC) and the like or a mixture of the listed solvents. Accordingly, the reagents used in the present invention for removal of the protection group comprises 15% to 25% of the organic base prepared in an organic solvent.
  • the organic base is prepared in an organic solvent is employed in the deprotection of the bound peptide chain to the resin.
  • the protected peptide is cleaved from the peptide resin and deprotected, simultaneously to obtain linear Abaloparatide.
  • the present process for solid phase synthesis provides deprotection of the peptide using a combination of Trifluoroacetic acid (TFA) and radical scavengers.
  • TFA Trifluoroacetic acid
  • one or more radical scavengers are selected from the group comprising triisopropylsilane (TIS), dithiothreitol (DTT), 1,2- ethanedithiol (EDT), Phenol, cresol, thioanisole, ammonium iodide, DMS and water.
  • TIS triisopropylsilane
  • DTT dithiothreitol
  • EDT 1,2- ethanedithiol
  • Phenol Phenol
  • cresol thioanisole
  • ammonium iodide DMS and water.
  • the column which may be used for the reverse-phase chromatography may be any known column employed in the art.
  • the column may be C-8 or C-18 column.
  • Abaloparatide was purified by reverse phase C-8 HPLC using a combination of a buffer and an organic solvent. More preferably, the said combination comprises triethylammonium phosphate (TEAP) buffer and Acetonitrile. Post purification, the fractions were analysed for their purity. The fraction containing Abaloparatide having >97% were pooled and were further taken up for salt exchange (desalting). The salt exchange was performed on RP-HPLC using ammonium acetate.
  • TEAP triethylammonium phosphate
  • the present invention provides a substantially pure Abaloparatide having Seq Id No.1 free from impurities of peptide aggregates.
  • substantially pure refers to chemical and optical purity of greater than about 99%.
  • the yield of crude Abaloparatide obtained in the present invention is 100%.
  • the overall isolated purification yield is in the range of 22% to 30%.
  • the present invention provides a formulation comprising Abaloparatide and one or more pharmaceutically acceptable excipients.
  • the present invention provides Abaloparatide prepared according to the process of the present invention for use in the treatment of osteoporosis and for postmenopausal women with osteoporosis who are more susceptible to bone fractures. Accordingly, there is provided herein a method for treating osteoporosis, the method comprising administering to a subject in need thereof a therapeutically effective amount of Abaloparatide prepared according to the process of the present invention.
  • the present process for preparation of Abaloparatide having Seq Id No.1 which is a parathyroid hormone can be applied with suitable modifications in amino acid groups, reagents and process parameters to the synthesis of other human parathyroid hormones.
  • the following examples, which include preferred embodiments, will serve to illustrate the practice of this invention, it being understood that the particulars shown are by way of example and for purpose of illustrative discussion of preferred embodiments of the invention. Examples: Example 1:
  • Fmoc-Rink amide MBHA resin with a Loading of ⁇ 0.33 mmol/gram (about 30.3 gm resin, 10 mmol) was swelled in DMF for 30 mins by agitation under nitrogen, decanting the solvent, washing the resin twice by using DMF.
  • Fmoc-deprotection of the Rink amide resin was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF.
  • the coupling of the first amino acid Fmoc-Ala-OH was carried out by addition of HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.
  • Stage-2 Synthesis of Fmoc-Thr(tbu) 33 - Ala 34 –Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF.
  • the Fmoc-Thr(tbu)-OH (25 mmole, 2.5eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT.
  • Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20% piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF.
  • the Fmoc-His(Trt)-OH (25 mmole, 2.5eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent.
  • the mixture was stirred in the presence of Nitrogen for 40-50 mins at RT.
  • the excess reagents were drained and washed the peptidyl resin four times by DMF.
  • Stage-4 Synthesis of Fmoc-Leu 31 -His(Trt) 32 -Thr(tbu) 33 -Ala 34 –Rink amide MBHA Resin
  • Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF.
  • the Fmoc-Leu-OH (25 mmole, 2.5eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT.
  • Stage-5 Synthesis of Fmoc-Lys(boc) 30 -Leu 31 -His(Trt) 32 -Thr(tbu) 33 -Ala 34 – Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20% piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF.
  • the Fmoc- Lys(boc)-OH (25 mmole, 2.5eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.
  • Stage-6 Synthesis of Fmoc-Leu 28 -Aib 29 -Lys(boc) 30 -Leu 31 -His(Trt) 32 - Thr(tbu) 33 - Ala 34 –Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Leu-Aib-OH dipeptide (25 mmole, 2.5eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent.
  • Stage-7 Synthesis of Fmoc-Leu 27 -Leu 28 -Aib 29 -Lys(boc) 30 -Leu 31 -His(Trt) 32 - Thr(tbu) 33 - Ala 34 –Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF.
  • the Fmoc-Leu-OH (25 mmole, 2.5eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.
  • Stage-8 Synthesis of Fmoc-Lys(boc) 26 -Leu 27 -Leu 28 -Aib 29 -Lys(boc) 30 -Leu 31 - His(Trt) 32 -Thr(tbu) 33 - Ala 34 –Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF.
  • the Fmoc-Lys(boc)-OH (25 mmole, 2.5eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.
  • Stage-9 Synthesis of Fmoc-Glu(tbu) 25 -Lys(boc) 26 -Leu 27 -Leu 28 -Aib 29 - Lys(boc) 30 -Leu 31 -His(Trt) 32 -Thr(tbu) 33 - Ala 34 –Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF.
  • the Fmoc-Glu(tbu)-OH (25 mmole, 2.5eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.
  • Stage-10 Synthesis of Fmoc-Leu 24 -Glu(tbu) 25 -Lys(boc) 26 -Leu 27 -Leu 28 -Aib 29 - Lys(boc) 30 -Leu 31 -His(Trt) 32 -Thr(tbu) 33 - Ala 34 –Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF.
  • the Fmoc-Leu-OH (25 mmole, 2.5eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.
  • Stage-11 Synthesis of Fmoc-Leu 23 -Leu 24 -Glu(tbu) 25 -Lys(boc) 26 -Leu 27 -Leu 28 - Aib 29 -Lys(boc) 30 -Leu 31 -His(Trt) 32 -Thr(tbu) 33 -Ala 34 –Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF.
  • the Fmoc-Leu-OH (25 mmole, 2.5eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.
  • Stage-12 Synthesis of Fmoc-Glu(tbu) 22 -Leu 23 -Leu 24 -Glu(tbu) 25 -Lys(boc) 26 - Leu 27 -Leu 28 -Aib 29 -Lys(boc) 30 -Leu 31 -His(Trt) 32 -Thr(tbu) 33 -Ala 34 –Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20% piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF.
  • the Fmoc-Glu(tbu)-OH (25 mmole, 2.5eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.
  • Stage-13 Synthesis of Fmoc-Arg(pbf) 21 -Glu(tbu) 22 -Leu 23 -Leu 24 -Glu(tbu) 25 - Lys(boc) 26 -Leu 27 -Leu 28 -Aib 29 -Lys(boc) 30 -Leu 31 -His(Trt) 32 -Thr(tbu) 33 - Ala 34 – Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF.
  • the Fmoc- Arg(pbf)-OH (25 mmole, 2.5eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.
  • Stage-14 Synthesis of Fmoc-Arg(pbf) 20 -Arg(pbf) 21 -Glu(tbu) 22 -Leu 23 -Leu 24 - Glu(tbu) 25 -Lys(boc) 26 -Leu 27 -Leu 28 -Aib 29 -Lys(boc) 30 -Leu 31 -His(Trt) 32 - Thr(tbu) 33 - Ala 34 –Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF.
  • the Fmoc-Arg(pbf)-OH (25 mmole, 2.5eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.
  • Stage-15 Synthesis of Fmoc-Arg(pbf) 19 -Arg(pbf) 20 -Arg(pbf) 21 -Glu(tbu) 22 - Leu 23 -Leu 24 -Glu(tbu) 25 -Lys(boc) 26 -Leu 27 -Leu 28 -Aib 29 -Lys(boc) 30 -Leu 31 - His(Trt) 32 -Thr(tbu) 33 -Ala 34 –Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF.
  • the Fmoc-Arg(pbf)-OH (25 mmole, 2.5eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.
  • Stage-16 Synthesis of Fmoc-Leu 18 -Arg(pbf) 19 -Arg(pbf) 20 -Arg(pbf) 21 - Glu(tbu) 22 -Leu 23 -Leu 24 -Glu(tbu) 25 -Lys(boc) 26 -Leu 27 -Leu 28 -Aib 29 -Lys(boc) 30 - Leu 31 -His(Trt) 32 -Thr(tbu) 33 - Ala 34 –Rink amide MBHA Resin: Fmoc- deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF.
  • the Fmoc-Leu-OH (25 mmole, 2.5eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.
  • Stage-17 Synthesis of Fmoc-Asp(tbu) 17 -Leu 18 -Arg(pbf) 19 -Arg(pbf) 20 - Arg(pbf) 21 -Glu(tbu) 22 -Leu 23 -Leu 24 -Glu(tbu) 25 -Lys(boc) 26 -Leu 27 -Leu 28 -Aib 29 - Lys(boc) 30 -Leu 31 -His (Trt) 32 -Thr(tbu) 33 -Ala 34 –Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF.
  • the Fmoc-Asp(tbu)-OH (25 mmole, 2.5eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.
  • Stage-18 Synthesis of Fmoc-Gln 16 -Asp(tbu) 17 -Leu 18 -Arg(pbf) 19 -Arg(pbf) 20 - Arg(pbf) 21 -Glu(tbu) 22 -Leu 23 -Leu 24 -Glu(tbu) 25 -Lys(boc) 26 -Leu 27 -Leu 28 -Aib 29 - Lys(boc) 30 -Leu 31 -His(Trt) 32 -Thr(tbu) 33 -Ala 34 –Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF.
  • the Fmoc-Gln-OH (25 mmole, 2.5eq.) was coupled using HOBt (50 mmole, 5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.
  • Stage-19 Synthesis of Fmoc-Ile 15 -Gln 16 -Asp(tbu) 17 -Leu 18 -Arg(pbf) 19 - Arg(pbf) 20 -Arg(pbf) 21 -Glu(tbu) 22 -Leu 23 -Leu 24 -Glu(tbu) 25 -Lys(boc) 26 -Leu 27 - Leu 28 -Aib 29 -Lys(boc) 30 -Leu 31 -His(Trt) 32 -Thr(tbu) 33 -Ala 34 –Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF.
  • the Fmoc-Ile-OH (25 mmole, 2.5eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.
  • Stage-20 Synthesis of Fmoc-Lys(boc) 13 -Ser[Psi(Me,Me)pro] 14 -Ile 15 -Gln 16 - Asp(tbu) 17 -Leu 18 -Arg(pbf) 19 -Arg(pbf) 20 -Arg(pbf) 21 -Glu(tbu) 22 -Leu 23 -Leu 24 - Glu(tbu) 25 -Lys(boc) 26 -Leu 27 -Leu 28 -Aib 29 -Lys(boc) 30 -Leu 31 -His(Trt) 32 - Thr(tbu) 33 -Ala 34 –Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF.
  • the Fmoc- 13 Lys(boc)- 14 Ser[Psi(Me,Me)pro]-OH (20 mmole, 2 eq.) was coupled using HOBt (20 mmole, 2 eq) and DIC (20 mmole, 2 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.
  • the Fmoc-Gly-OH (25 mmole, 2.5 eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.
  • Stage-22 Synthesis of Fmoc-Lys(boc) 11 -Gly 12 -Lys(boc) 13 - Ser[Psi(Me,Me)pro] 14 -Ile 15 -Gln 16 -Asp(tbu) 17 -Leu 18 -Arg(pbf) 19 -Arg(pbf) 20 - Arg(pbf) 21 -Glu(tbu) 22 -Leu 23 -Leu 24 -Glu(tbu) 25 -Lys(boc) 26 -Leu 27 -Leu 28 -Aib 29 - Lys(boc) 30 -Leu 31 -His(Trt) 32 -Thr(tbu) 33 -Ala 34 –Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF.
  • the Fmoc-Lys(boc)-OH (25 mmole, 2.5 eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.
  • Stage-23 Synthesis of Fmoc-Asp(tbu) 10 -Lys(boc) 11 -Gly 12 -Lys(boc) 13 - Ser[Psi(Me,Me) pro] 14 -Ile 15 -Gln 16 -Asp(tbu) 17 -Leu 18 -Arg(pbf) 19 -Arg(pbf) 20 - Arg(pbf) 21 -Glu(tbu) 22 -Leu 23 -Leu 24 -Glu(tbu) 25 -Lys(boc) 26 -Leu 27 -Leu 28 -Aib 29 - Lys(boc) 30 -Leu 31 -His(Trt) 32 -Thr(tbu) 33 -Ala 34 –Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing
  • the Fmoc-Asp(tbu)-OH (25 mmole, 2.5 eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.
  • the Fmoc-His(Trt)-OH (25 mmole, 2.5 eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.
  • the Fmoc-Leu-OH (25 mmole, 2.5 eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.
  • Stage-26 Synthesis of Fmoc-Leu 7 -Leu 8 -His(Trt) 9 -Asp(tbu) 10 -Lys(boc) 11 - Gly 12 -Lys(boc) 13 -Ser[Psi(Me,Me)pro] 14 -Ile 15 -Gln 16 -Asp(tbu) 17 -Leu 18 - Arg(pbf) 19 -Arg(pbf) 20 -Arg(pbf) 21 -Glu(tbu) 22 -Leu 23 -Leu 24 -Glu(tbu) 25 - Lys(boc) 26 -Leu 27 -Leu 28 -Aib 29 -Lys(boc) 30 -Leu 31 -His(Trt) 32 -Thr(tbu) 33 -Ala 34 – Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin
  • the Fmoc-Leu- OH (25 mmole, 2.5 eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.
  • the Fmoc-Gln- OH (25 mmole, 2.5 eq.) was coupled using HOBt (50 mmole, 5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.
  • Stage-28 Synthesis of Fmoc-His(Trt) 5 -Gln 6 -Leu 7 -Leu 8 -His(Trt) 9 -Asp(tbu) 10 - Lys(boc) 11 -Gly 12 -Lys(boc) 13 -Ser[Psi(Me,Me)pro] 14 -Ile 15 -Gln 16 -Asp(tbu) 17 - Leu 18 -Arg(pbf) 19 -Arg(pbf) 20 -Arg(pbf) 21 -Glu(tbu) 22 -Leu 23 -Leu 24 -Glu(tbu) 25 - Lys(boc) 26 -Leu 27 -Leu 28 -Aib 29 -Lys(boc) 30 -Leu 31 -His(Trt) 32 -Thr(tbu) 33 -Ala 34 – Rink amide MBHA Resin: Fmoc-deprotect
  • the Fmoc- His(Trt)-OH (25 mmole, 2.5 eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.
  • Stage-29 Synthesis of Fmoc-Glu(tbu) 4 -His(Trt) 5 -Gln 6 -Leu 7 -Leu 8 -His(Trt) 9 - Asp(tbu) 10 -Lys(boc) 11 -Gly 12 -Lys(boc) 13 -Ser[Psi(Me,Me)pro] 14 -Ile 15 -Gln 16 - Asp(tbu) 17 -Leu 18 -Arg(pbf) 19 -Arg(pbf) 20 -Arg(pbf) 21 -Glu(tbu) 22 -Leu 23 -Leu 24 - Glu(tbu) 25 -Lys(boc) 26 -Leu 27 -Leu 28 -Aib 29 -Lys(boc) 30 -Leu 31 -His(Trt) 32 - Thr(tbu) 33 -Ala 34 –Rink amide MBHA
  • the Fmoc-Glu(tbu)-OH (25 mmole, 2.5 eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.
  • the Fmoc-Ser(tbu)-OH (25 mmole, 2.5 eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.
  • Stage-31 Synthesis of Fmoc-Val 2 -Ser(tbu) 3 -Glu(tbu) 4 -His(Trt) 5 -Gln 6 -Leu 7 - Leu 8 -His(Trt) 9 -Asp(tbu) 10 -Lys(boc) 11 -Gly 12 -Lys(boc) 13 -Ser[Psi(Me,Me)pro] 14 - Ile 15 -Gln 16 -Asp(tbu) 17 -Leu 18 -Arg(pbf) 19 -Arg(pbf) 20 -Arg(pbf) 21 -Glu(tbu) 22 - Leu 23 -Leu 24 -Glu(tbu) 25 -Lys(boc) 26 -Leu 27 -Leu 28 -Aib 29 -Lys(boc) 30 -Leu 31 - His(Trt) 32 -Thr(tbu) 33 -
  • the Fmoc-Val-OH (25 mmole, 2.5 eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.
  • Stage-32 Synthesis of Fmoc-Ala 1 -Val 2 -Ser(tbu) 3 -Glu(tbu) 4 -His(Trt) 5 -Gln 6 - Leu 7 -Leu 8 -His(Trt) 9 -Asp(tbu) 10 -Lys(boc) 11 -Gly 12 -Lys(boc) 13 - Ser[Psi(Me,Me)pro] 14 -Ile 15 -Gln 16 -Asp(tbu) 17 -Leu 18 -Arg(pbf) 19 -Arg(pbf) 20 - Arg(pbf) 21 -Glu(tbu) 22 -Leu 23 -Leu 24 -Glu(tbu) 25 -Lys(boc) 26 -Leu 27 -Leu 28 -Aib 29 - Lys(boc) 30 -Leu 31 -His(Trt) 32 -Thr(t
  • the Fmoc-Ala-OH (25 mmole, 2.5 eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.
  • Stage-33 Synthesis of H-Ala 1 -Val 2 -Ser(tbu) 3 -Glu(tbu) 4 -His(Trt) 5 -Gln 6 -Leu 7 - Leu 8 -His(Trt) 9 -Asp(tbu) 10 -Lys(boc) 11 -Gly 12 -Lys(boc) 13 -Ser[Psi(Me,Me)pro] 14 - Ile 15 -Gln 16 -Asp(tbu) 17 -Leu 18 -Arg(pbf) 19 -Arg(pbf) 20 -Arg(pbf) 21 -Glu(tbu) 22 - Leu 23 -Leu 24 -Glu(tbu) 25 -Lys(boc) 26 -Leu 27 -Leu 28 -Aib 29 -Lys(boc) 30 -Leu 31 - His(Trt) 32 -Thr(tbu) 33
  • the cleavage was carried out at ⁇ 10°C for initial 15 minutes followed by the stirring of the peptidyl resin for 3 hours at ambient temperature.
  • the crude cleavage mixture was then filtered, the resin washed thoroughly with TFA.
  • the filtrate was dropped on to 12 mL of cold dry Diethyl ether per mL of cocktail and further 6 additional washing with 0.5 Liter of diethyl ether were done to the product.
  • Product was dried under vacuum for 16 hrs.
  • Example 3 Preparation method of Abaloparatide linear peptide resin on rink amide resin of functionality 0.52 mmol/g.
  • Crude Abaloparatide was purified by reverse phase C-8 HPLC using TEAP buffer (as buffer A) and Acetonitrile (as buffer B). Post purification, the fractions were analysed for the purity. The fraction containing >97% were pooled and were further taken up for salt exchange (desalting). The salt exchange was performed on RP-HPLC using ammonium acetate. The fractions containing >99% were pooled and were lyophilized to isolate pure Abaloparatide which is further stored at -20°C.
  • Example 5 Preparation method of Abaloparatide linear peptide resin on rink amide resin of functionality 0.33 mmol/g (without using pseudoproline Fmoc-Lys(Boc) Ser[Psi(Me,Me)Pro]-OH unit): The process is same as described in the Example 1, and the only change is that “No pseudoproline dipeptide unit is employed in the synthesis”. (Process yield 80 %, purity 37 %).HPLC chromatogram of Crude Abaloparatide acetate is depicted in FIG 6. Advantages of the present invention:
  • Fmoc-Leu-Aib-OH reduces possible deletion impurities which can be formed due to incomplete coupling of Fmoc-Leu28-OH after Fmoc-Aib29- OH Coupling to the peptide chain bound to the resin in SPPS.

Abstract

The present invention relates to a process for preparing Abaloparatide by The Solid Phase Process Synthesis (SPPS). The said process employs protected dipeptide units thereby avoids issues related to aggregation of peptides on to the solid support and racemization, therefore rendering a high purity of the resultant Abaloparatide.

Description

“A PROCESS FOR PREPARING ABALOPARATIDE”
TECHNICAL FIELD OF THE INVENTION:
The present invention relates to an improved process for the preparation of Abaloparatide having Seq Id No.1.
BACKGROUND AND PRIOR ART OF THE INVENTION:
Osteoporosis in postmenopausal women represents a disease burden for which diagnosis and treatment should be healthcare priorities. An osteoporotic fracture can be a life-altering event for a woman and her family.
Abaloparatide is an analog of parathyroid hormone related protein ( PTHrP ) and is a synthetic 34 amino acid peptide. It was approved in 2017 by the FDA (marketed under the trade name Tymlos) for the treatment of postmenopausal women with osteoporosis at a high risk for fracture. Abaloparatide is a synthetic peptide that is related to human PTHrP ( hPTHrP ) and its potential has been demonstrated in preclinical testing to widen the anabolic window for bone therapeutics, stimulating bone formation with a limited effect on bone resorption and mineral mobilization. This could enable improved convenience over currently available anabolic therapies, resulting in greater compliance and, ultimately, greater benefit to patients.
The aggregation of peptide chains in the process for Solid phase peptide synthesis (SPPS) has been a challenging issue faced by researchers in the synthesis of long chain peptides. A successful peptide assembly of long peptides by SPPS is hampered by problems associated with incomplete couplings and de-protections resulting in inferior yields and homogeneity of peptide products. Inter- and intramolecular hydrogen bonding resulting in the aggregation of peptide chains to form secondary structures, such as b-sheet formation, is now thought to be a major cause of problems associated with synthesis in SPPS. Various strategies have been advocated to circumvent the problem of aggregation in Fmoc SPPS and to overcome the difficulties in long peptide chain synthesis, including solvent composition, elevated temperatures, use of chaotropic salts or solubilizing protecting group. US Patent No. 6,921,750 discloses a process for the synthesis of human parathyroid hormones, viz. Abaloparatide using Boc-SPPS methodology which requires the addition of hazardous hydrofluoric acid (HF) cleavage. However, the use of toxic HF is hazardous and is therefore not suitable for large scale industrial application in the preparation of Abaloparatide. The ease of assembly of a given sequence is generally hard to predict and is one of the factors that makes peptide synthesis interesting and challenging, although peptides containing stretches of contiguous hydrophobic amino acids like Ala, Val, Ile as well as those containing amino acids which can form intra-chain hydrogen bonds, such as Gln, Ser and Thr, are frequently difficult to make. For this reason, it is generally better to adopt from the outset synthetic strategies that mitigate the effects of structure formation. For peptide sequences longer than 20 amino acids it is therefore strongly recommended to monitor the peptide assembly by small TFA cleavages. Chinese Patent Publication No. 106146648 discloses synthesis of Abaloparatide using three separately synthesized fragment. CN 106146648 discloses a process to prepare parathyroid hormone analogue (Abaloparatide) by synthesizing separately three fragments (1-15) and (16-23) and (24-33) and then coupling the said fragments to obtain the Abaloparatide. This Chinese patent application makes use of three separately synthesized fragments on three different resins which increase the cost of the entire process. The use of different resins in the synthesis of Abaloparatide fragments causes the process to be tedious and inconvenient. Additionally, the process disclosed therein is prone to isomerization leading to optically impure Abaloparatide which is difficult to purify due to its homogeneity. Therefore, there is a need in the art to arrive at a process that is suitable for the synthesis long peptide chains without issues of aggregation and having an enhanced purity of the crude Abaloparatide being synthesized by the said process. OBJECT OF THE INVENTION:
It is an object of the present invention to provide an improved solid phase peptide synthesis process for the preparation of Abaloparatide. It is another object of the present invention to provide a process for the synthesis of long chain peptides such that the said process overcomes issues relating to aggregation of peptide chains on to the resins, thereby obtaining higher yield and better purity of Abaloparatide. It is yet another object of the present invention to provide a process for solid phase peptide synthesis for preparation of Abaloparatide by employing protected dipeptide units and amino acids. SUMMARY OF THE INVENTION:
In an aspect, the present invention provides a process for the synthesis of Abaloparatide represented by Seq Id No.1 comprising;
(i) elongation of a peptide with sequential addition of protected amino acid(s) and dipeptide units by solid phase synthesis to a solid support to obtain a protected peptide bound to a support,
(ii) cleaving and de-protecting the resin simultaneously to obtain crude Abaloparatide followed by purification to obtain purified Abaloparatide; and (iii) isolation of Abaloparatide. wherein the a-amino group of the amino acid/dipeptide is protected by Fmoc, wherein a peptide bond is formed between a carboxyl group of the Fmoc protected amino acid/dipeptide unit and the amino group linked to the support. According to an advantageous aspect of the present invention there is provided an improved process for preparing Abaloparatide, wherein the disadvantage of peptide aggregation on to the solid support as observed in the conventional processes of solid phase synthesis for long chain peptides is overcome by involving dipeptide units. In another aspect of the present invention there is provided Abaloparatide prepared according to the process of the present invention having purity of 80% to 85% prior to the purification step and >99% purity after the purification step. In yet another aspect of the present invention there is provided a pharmaceutical composition comprising Abaloparatide prepared according to the process of the present invention together with one or more pharmaceutically acceptable excipients. DETAILED DESCRIPTION OF DRAWINGS OF THE INVENTION:
Figure 1 depicts the scheme for the solid phase peptide synthesis (SPPS) for Abaloparatide
Figure 2 depicts the HPLC chromatogram of Crude Abaloparatide obtained by the process of the present invention;
Figure 3 depicts the HPLC chromatogram for % purity of Abaloparatide synthesized on use of rink amide resin with the standard substitution value of 0.50-0.60 mmol/gm;
Figure 4 depicts the HPLC chromatogram of purified Abaloparatide obtained by the present invention;
Figure 5 depicts the mass of purified Abaloparatide thereby confirming the formation of Abaloparatide;
Figure 6 depicts the purity of crude Abaloparatide obtained by in the absence of Fmoc-Lys(Boc) Ser[Psi(Me,Me)Pro]-OH unit. ABBREVIATIONS:
ACN: acetonitrile
Aib: 2-Methylalanine
Ala: alanine
Arg: arginine
Asn: asparagine
Asp: aspartic acid.
Boc: t-butyloxycarbonyl
DCM; dichloromethane
DIC: Diisopropylcarbodiimide
DIPEA: Diisopropylethylamine
DMF: N,N-dimethylformamide
DMS: Dimethyl sulfide
DTT: dithiothreitol
EDT: 1,2-ethanedithiol
eq: equivalents
Fmoc: 9-fluorenylmethoxycarbonyl
Gln: glutamine
Glu: glutamic acid
Gly: glycine
His: histidine
HOBt: N-hydroxybenzotriazole
HPLC: high performance liquid chromatography Ile: isoleucine
Leu: leucine
Lys: lysine
MBHA resin: Methylbenzylhydrylamine resin
MeOH: methanol
NMM: N-methyl morpholine
OtBu: t-butyl ester
Pbf: 2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl Phe: phenylalanine
RT: room or ambient temperature;
Ser: serine
SPPS: Solid Phase Peptide Synthesis
tBu: t-butyl
TFA: trifluoroacetic acid
THF: Tetrahydrofuran
TIPS, TIS: triisopropylsilane
TMS-Cl: Trimethylsilyl chloride
Trp: tryptophan
Trt: trityl or triphenylmethyl
Tyr: tyrosine
Val: valine DETAILED DESCRIPTION OF THE INVENTION:
The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated. The present invention relates to a process for preparing Abaloparatide by elongation of Fmoc protected fragments, simultaneous cleavage and de-protection of the peptide chain, purification and isolation of Abaloparatide. In a preferred embodiment, the present invention provides a process for the synthesis of Abaloparatide represented by Seq Id No.1 comprising;
(i) elongation of a peptide with sequential addition of protected amino acid(s) and dipeptide units by solid phase synthesis to a solid support to obtain a protected peptide bound to a support,
(ii) cleaving and de-protecting the resin simultaneously to obtain crude Abaloparatide followed by purification to obtain purified Abaloparatide; and (iii) isolation of Abaloparatide; wherein the a-amino group of the amino acid/dipeptide is protected by Fmoc,
wherein a peptide bond is formed between a carboxyl group of the Fmoc protected amino acid/dipeptide unit and the amino group lined to the support. Accordingly, Abaloparatide is synthesized by solid-phase peptide synthesis (SPPS). Keeping in line with the present invention, SPPS can be defined as a process in which a dipeptide unit or amino acid anchored by its C-terminus to a resin is assembled by the successive addition of the said protected amino acids constituting its sequence. In an embodiment, for SPPS the protecting group is selected from the group consisting of traditional Fmoc/tBu protection or Boc/benzyl protection. Other protecting groups such as Cbz, Bpoc could also be used as amino protecting group. More preferably, the protecting group for the amino acid/dipeptide unit is Fmoc. In another preferred embodiment, the present invention provides Fmoc protected amino acid selected from the group comprising Fmoc-Ala-OH; Fmoc-Thr(tbu)- OH; Fmoc-His(Trt)-OH; Fmoc-Leu-OH; Fmoc-Lys(Boc)-OH, Fmoc-Leu-Aib- OH optionally for position (28) and (29); Fmoc-Glu(tbu)-OH; Fmoc-Arg(Pbf)- OH; Fmoc-Asp(OtBu)-OH; Fmoc-Gln-OH; Fmoc-Ile-OH; Fmoc-Lys(boc)- Ser[Psi(Me,Me)Pro]-OH optionally for position (13) and (14); Fmoc-Gly-OH; Fmoc-Ser(tbu)-OH; Fmoc-Val-OH. In another preferred embodiment the present invention provides Fmoc-Lys(boc)- Ser[Psi(Me,Me)Pro]-OH is a Dimethyloxazolidine dipeptide. Dimethyloxazolidine dipeptides introduce into the peptide chain a pseudoproline residue, derived from either Ser or Thr, which disrupts peptide chain aggregation in the same manner as proline. In a further preferred embodiment, the present invention used Fmoc-Leu-Aib-OH dipeptide unit to avoid the possible deletion impurities which can be formed due to incomplete coupling of Fmoc-Leu28-OH after Fmoc-Aib29-OH Coupling to the peptide chain bound to the rink amide resin in SPPS. The use of Fmoc-Leu-Aib-OH in the present process is to overcome the difficulty observed in the detection of Fmoc removal of Fmoc-Aib29- from the peptide chain bound to the resin in the synthesis of Abaloparatide. After coupling of Fmoc-Aib29-OH on the growing chain of Abaloparatide during the Solid phase, it has been observed that after the deprotection of N-Fmoc group of Fmoc-Aib29- OH by 20% Piperidine/DMF mixture, Kaiser test was not positive i.e. resin beads were not showing expected blue colour instead they showed yellow colour with clear solution (-ive test). Most probable reason is supposed to be due to the steric hindrance of the methyl groups on the a carbon of 2-Methylalanine (Aib) group which probably shields the NH2 group of H-Aib29-OH for Kaiser Test. To overcome this problem, the present inventors have synthesized Fmoc-Leu-AIB- OH dipeptide by solution phase and introduced the same in the process for Abaloparatide solid phase synthesis After insertion of Fmoc-Leu-Aib-OH into the peptide chain, the deprotection of N-Fmoc group of Fmoc-leu28-OH by 20% Piperidine/DMF mixture the Kaiser test was as expected (+ive). It is worth to mention here that the possibility of racemization while coupling of Fmoc-Leu-Aib-OH to the peptide chain bound to the resin in SPPS is minimum due to the unavailability of a-Hydrogen in the Aib residue. In an embodiment of the present invention the solid support is a resin, wherein the said resin is preferably a rink amide resin. The said rink amide resin is selected from the group comprising 4-methylbenzhydrylamine resin (MBHA), Rink amide BHA resin or Sieber resin., preferably Rink amide MBHA resin. The 4-methylbenzhydrylamine resin (MBHA) is the most preferable resin being used having a low substitution value of 0.1 to 0.4 mmol/g. The synthesis of Abaloparatide when performed on rink amide resin of standard substitution value of 0.50-0.60 mmole/g affords the crude Abaloparatide of very low quality of approximately 45% purity, however the use of rink amide resin of low substitution value of 0.1-0.4 mmol/gm in the present invention affords the crude Abaloparatide having purity in a range of 80% to 85%. Figure 3 depicts the substantial low purity of crude Abaloparatide in the event of using of a rink amide resin with a standard substitution value. In an embodiment of the present invention the coupling agents in the process is selected from the group comprising hydroxybenzotriazole (HOBt), N, N'- diisopropylcarbodiimide (DIC), O-(Benzotriazol-1-yl)-N,N,N',N'- tetramethyluronium tetrafluoroborate (TBTU), N,N,N¢,N¢-Tetramethyl-O-(1H- benzotriazol-1-yl)uronium hexafluorophosphate (HBTU), 1,3- dicyclohexylcarbodlimide (DCC), 1-(dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC HCl), benzotriazol-1-yl-oxy-tris(dimethyl-amino)- phosphonium hexafluorophosphate (BOP), N,N-bis-(2-oxo-3- oxazolidinyl)phosphonic dichloride (BOP-C1), benzotriazol-1- yloxytri(pyrrolidino)phosphonium hexafluorophosphate (PyBOP), bromotri(pyrrolidino)phosphonium hexafluorophosphate (PyBrOP), chlorotri(pynolidino)phosphonium hexafluorophosphate (PyClOP), ethyl-2- cyano-2-(hydroxyimino) acetate (Oxyma Pure), O-(6-Chloro-1- hydrocibenzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TCTU), 245-norbornen-2,3-dicarboximido)-1,1,3,3-tetramethyluronium tetrafluoroborate (TNTU), 2-succinimido-1,1,3,3-tetramethyluronium tetrafluoro borate (TSTU), 1- Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU), 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin- 4(3H)-one (DEPBT) or mixtures thereof. In an embodiment of the present invention the coupling takes place in presence of a solvent selected from the group comprising of DMF, DCM, THF, NMP, DMAC methanol, ethanol, isopropanol, dichloroethane, 1 ,4- dioxane, 2-methyl tetrahydrofuran ethyl acetate, acetonitrile, acetone, and the like or a mixtures thereof. Further, Fmoc is removed from the peptide in the presence of an organic base prepared in an organic solvent. The organic base is selected from the group comprising piperidine, piperazine, N-methyl morpholine, diethyl amine, triethyl amine, 1,8-Diazabicyclo [5.4.0]undec-7-ene (DBU) and the like or a mixtures thereof. The organic solvent is selected from the group comprising dimethyl formamide (DMF), N-Methyl-2-Pyrrolidone (NMP), dichloromethane (DCM), tetrahydrofuran (THF), N,N-dimethylacetamide (DMAC) and the like or a mixture of the listed solvents. Accordingly, the reagents used in the present invention for removal of the protection group comprises 15% to 25% of the organic base prepared in an organic solvent. Preferably, 20% of the organic base is prepared in an organic solvent is employed in the deprotection of the bound peptide chain to the resin. In an embodiment, the protected peptide is cleaved from the peptide resin and deprotected, simultaneously to obtain linear Abaloparatide. In an embodiment, the present process for solid phase synthesis provides deprotection of the peptide using a combination of Trifluoroacetic acid (TFA) and radical scavengers. Accordingly, one or more radical scavengers are selected from the group comprising triisopropylsilane (TIS), dithiothreitol (DTT), 1,2- ethanedithiol (EDT), Phenol, cresol, thioanisole, ammonium iodide, DMS and water. Simultaneous deprotection of all the protecting groups was carried out by the treatment of either of the cocktail cleavage mixtures stated below: TFA:EDT:Phenol:Thioanisole:water (82.5:2.5:5:5:5 v/v) or TFA:TIS:DTT:Water (85:5:5:5 v/v) preferably with TFA:EDT:Phenol:Thioanisole:water (82.5:2.5:5:5:5 v/v) cocktail mixture i.e. K type reagent. The crude Abaloparatide obtained after cleaving and deptrotecting the peptide from the resin is further purified by Reverse Phase HPLC. The column which may be used for the reverse-phase chromatography may be any known column employed in the art. In one embodiment, the column may be C-8 or C-18 column. Abaloparatide was purified by reverse phase C-8 HPLC using a combination of a buffer and an organic solvent. More preferably, the said combination comprises triethylammonium phosphate (TEAP) buffer and Acetonitrile. Post purification, the fractions were analysed for their purity. The fraction containing Abaloparatide having >97% were pooled and were further taken up for salt exchange (desalting). The salt exchange was performed on RP-HPLC using ammonium acetate. The fractions containing Abaloparatide having >99% were pooled and were lyophilized to isolate pure Abaloparatide which is further stored at -20°C. In an embodiment, the present invention provides a substantially pure Abaloparatide having Seq Id No.1 free from impurities of peptide aggregates. As used herein, substantially pure refers to chemical and optical purity of greater than about 99%. Further, the yield of crude Abaloparatide obtained in the present invention is 100%. The overall isolated purification yield is in the range of 22% to 30%. In a further preferred embodiment, the present invention provides a formulation comprising Abaloparatide and one or more pharmaceutically acceptable excipients. In another embodiment, the present invention provides Abaloparatide prepared according to the process of the present invention for use in the treatment of osteoporosis and for postmenopausal women with osteoporosis who are more susceptible to bone fractures. Accordingly, there is provided herein a method for treating osteoporosis, the method comprising administering to a subject in need thereof a therapeutically effective amount of Abaloparatide prepared according to the process of the present invention. The present process for preparation of Abaloparatide having Seq Id No.1 which is a parathyroid hormone can be applied with suitable modifications in amino acid groups, reagents and process parameters to the synthesis of other human parathyroid hormones. The following examples, which include preferred embodiments, will serve to illustrate the practice of this invention, it being understood that the particulars shown are by way of example and for purpose of illustrative discussion of preferred embodiments of the invention. Examples: Example 1:
Synthesis of Fmoc-Leu-Aib-OH dipeptide
To H-Aib-OH (43.76 gm, 424.44 mmol, 3 eq), dry THF (1400 ml) was added followed by addition of DIPEA (147.85 ml, 848.88 mmol, 6 eq) and TMS-Cl (107.36 ml, 848.88 mmol, 6 eq). Attached Reflux condenser to the RBF and reflux it for 2.5 hrs, followed by cooling it to RT and then at 5-10ºC for 1 hr (flask A). Meanwhile in the other RBF, The amino acid derivative, Fmoc-L-Leucine (50g, 141 mmol, 1 eq) was dissolved in dry THF (750 ml) and cooled to 0-5ºC, to that added NMM (17.84 ml, 155.62 mmol, 1.1 eq) followed by slow addition of Isobutyl chloroformate (36.8 ml, 282.96 mmol, 2 eq) while maintaining the temp at 0-5ºC, the stirring was continued in cooling condition for 1 hr (flask B). When the Stirring of flask B completed, Solution from Flask A slowly transferred in portions in to flask B with further rinsing with THF. Stirred the Reaction mixture in cooling conditions for 10 mins and then at RT. Reaction mixture was monitored by HPLC and taken for workup after consumption of starting material, the reaction mixture was worked up after 18 hr by evaporating the THF solvent under reduced pressure and re dissolving the obtained oil in 10% NaHCO3 Solution followed by washings with Diethyl ether. The phases were separated, and the aq. layer further extracted 3 times with Diethyl ether. Aq. Layer was further acidified with 10% HCl, sticky material obtained which is kept in walk in cool overnight and stirred further for 1 hr in cooling, Ppt formed was filtered, dried and further recrystallized with Hexane and Diethyl ether to give an 83% isolated yield of crystalline product after thorough drying under vacuum.
The isolated yield of the Fmoc-Leu-Aib-OH: 83% (51.8 gm)
The Purity of the crude Fmoc-Leu-Aib-OH: 97.5% Amino acids used: The SPPS was done with the following AA, if not stated otherwise: Fmoc-Ala-OH; Fmoc-Thr(tbu)-OH; Fmoc-His(Trt)-OH; Fmoc-Leu- OH; Fmoc-Lys(Boc)-OH Fmoc-Leu-Aib-OH optionally for position (28) and (29); Fmoc-Glu(tbu)-OH; Fmoc-Arg(Pbf)- OH; Fmoc-Asp(OtBu)-OH; Fmoc- Gln-OH; Fmoc-Ile-OH; Fmoc-Lys(boc)-Ser[Psi(Me,Me)Pro]-OH optionally for position (13) and (14); Fmoc-Gly-OH; Fmoc-Ser(tbu)-OH; Fmoc-Val-OH. The pseudoproline dipeptide, Fmoc-13Lys(boc)-14Ser[Psi(Me,Me)pro]-OH was incorporated using the standard synthesis cycle using 2.0x excess only. Additionally, dipeptide unit, Fmoc-28Leu-29Aib-OH was incorporated using the standard synthesis cycle using 2.5x excess only at position 28 and 29 of the 34mer Abaloparatide peptide. SPPS was done manually without an automatic synthesizer. Example 2: Preparation method of Abaloparatide linear peptide resin on rink amide resin of functionality 0.33 mmol/g low substitution value of 0.1 to 0.4 mmol/g)
The step wise process for preparation of Abaloparatide is depicted in Figure 1. Stage-1: Synthesis of Fmoc–Ala34–Rink amide MBHA Resin
Fmoc-Rink amide MBHA resin with a Loading of ~0.33 mmol/gram (about 30.3 gm resin, 10 mmol) was swelled in DMF for 30 mins by agitation under nitrogen, decanting the solvent, washing the resin twice by using DMF. Fmoc-deprotection of the Rink amide resin was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The coupling of the first amino acid Fmoc-Ala-OH (25 mmole, 2.5 eq), was carried out by addition of HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF. Stage-2: Synthesis of Fmoc-Thr(tbu) 33- Ala34–Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Thr(tbu)-OH (25 mmole, 2.5eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF. Stage-3: Synthesis of Fmoc-His(Trt)32-Thr(tbu) 33- Ala34–Rink amide MBHA Resin:
Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20% piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-His(Trt)-OH (25 mmole, 2.5eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF. Stage-4: Synthesis of Fmoc-Leu31-His(Trt)32-Thr(tbu)33-Ala34–Rink amide MBHA Resin Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Leu-OH (25 mmole, 2.5eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and the peptidyl resin was washed four times with DMF. Stage-5: Synthesis of Fmoc-Lys(boc)30-Leu31-His(Trt)32-Thr(tbu)33-Ala34– Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20% piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc- Lys(boc)-OH (25 mmole, 2.5eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF. Stage-6: Synthesis of Fmoc-Leu28-Aib29-Lys(boc)30-Leu31-His(Trt)32- Thr(tbu)33- Ala34–Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Leu-Aib-OH dipeptide (25 mmole, 2.5eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF. Stage-7: Synthesis of Fmoc-Leu27-Leu28-Aib29-Lys(boc)30-Leu31-His(Trt)32- Thr(tbu)33- Ala34–Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Leu-OH (25 mmole, 2.5eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF. Stage-8: Synthesis of Fmoc-Lys(boc)26-Leu27-Leu28-Aib29-Lys(boc)30-Leu31- His(Trt)32-Thr(tbu)33- Ala34–Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Lys(boc)-OH (25 mmole, 2.5eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF. Stage-9: Synthesis of Fmoc-Glu(tbu)25-Lys(boc)26-Leu27-Leu28-Aib29- Lys(boc)30-Leu31-His(Trt)32-Thr(tbu)33- Ala34–Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Glu(tbu)-OH (25 mmole, 2.5eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF. Stage-10: Synthesis of Fmoc-Leu24-Glu(tbu)25-Lys(boc)26-Leu27-Leu28-Aib29- Lys(boc)30-Leu31-His(Trt)32-Thr(tbu)33- Ala34–Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Leu-OH (25 mmole, 2.5eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF. Stage-11: Synthesis of Fmoc-Leu23-Leu24-Glu(tbu)25-Lys(boc)26-Leu27-Leu28- Aib29-Lys(boc)30-Leu31-His(Trt)32-Thr(tbu)33-Ala34–Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Leu-OH (25 mmole, 2.5eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF. Stage-12: Synthesis of Fmoc-Glu(tbu)22-Leu23-Leu24-Glu(tbu)25-Lys(boc)26- Leu27-Leu28-Aib29-Lys(boc)30-Leu31-His(Trt)32-Thr(tbu)33-Ala34–Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20% piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Glu(tbu)-OH (25 mmole, 2.5eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF. Stage-13: Synthesis of Fmoc-Arg(pbf)21-Glu(tbu)22-Leu23-Leu24-Glu(tbu)25- Lys(boc)26-Leu27-Leu28-Aib29-Lys(boc)30-Leu31-His(Trt)32-Thr(tbu)33- Ala34– Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc- Arg(pbf)-OH (25 mmole, 2.5eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF. Stage-14: Synthesis of Fmoc-Arg(pbf)20-Arg(pbf)21-Glu(tbu)22-Leu23-Leu24- Glu(tbu)25-Lys(boc)26-Leu27-Leu28-Aib29-Lys(boc)30-Leu31-His(Trt)32- Thr(tbu)33- Ala34–Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Arg(pbf)-OH (25 mmole, 2.5eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF. Stage-15: Synthesis of Fmoc-Arg(pbf)19-Arg(pbf)20-Arg(pbf)21-Glu(tbu)22- Leu23-Leu24-Glu(tbu)25-Lys(boc)26-Leu27-Leu28-Aib29-Lys(boc)30-Leu31- His(Trt)32-Thr(tbu)33-Ala34–Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Arg(pbf)-OH (25 mmole, 2.5eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF. Stage-16: Synthesis of Fmoc-Leu18-Arg(pbf)19-Arg(pbf)20-Arg(pbf)21- Glu(tbu)22-Leu23-Leu24-Glu(tbu)25-Lys(boc)26-Leu27-Leu28-Aib29-Lys(boc)30- Leu31-His(Trt)32-Thr(tbu)33- Ala34–Rink amide MBHA Resin: Fmoc- deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Leu-OH (25 mmole, 2.5eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF. Stage-17: Synthesis of Fmoc-Asp(tbu)17-Leu18-Arg(pbf)19-Arg(pbf)20- Arg(pbf)21-Glu(tbu)22-Leu23-Leu24-Glu(tbu)25-Lys(boc)26-Leu27-Leu28-Aib29- Lys(boc)30-Leu31-His (Trt)32-Thr(tbu)33-Ala34–Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Asp(tbu)-OH (25 mmole, 2.5eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF. Stage-18: Synthesis of Fmoc-Gln16-Asp(tbu)17-Leu18-Arg(pbf)19-Arg(pbf)20- Arg(pbf)21-Glu(tbu)22-Leu23-Leu24-Glu(tbu)25-Lys(boc)26-Leu27-Leu28-Aib29- Lys(boc)30-Leu31-His(Trt)32-Thr(tbu)33-Ala34–Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Gln-OH (25 mmole, 2.5eq.) was coupled using HOBt (50 mmole, 5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF. Stage-19: Synthesis of Fmoc-Ile15-Gln16-Asp(tbu)17-Leu18-Arg(pbf)19- Arg(pbf)20-Arg(pbf)21-Glu(tbu)22-Leu23-Leu24-Glu(tbu)25-Lys(boc)26-Leu27- Leu28-Aib29-Lys(boc)30-Leu31-His(Trt)32-Thr(tbu)33-Ala34–Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Ile-OH (25 mmole, 2.5eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF. Stage-20: Synthesis of Fmoc-Lys(boc) 13-Ser[Psi(Me,Me)pro] 14-Ile15-Gln16- Asp(tbu)17-Leu18-Arg(pbf)19-Arg(pbf)20-Arg(pbf)21-Glu(tbu)22-Leu23-Leu24- Glu(tbu)25-Lys(boc)26-Leu27-Leu28-Aib29-Lys(boc)30-Leu31-His(Trt)32- Thr(tbu)33-Ala34–Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-13Lys(boc)-14Ser[Psi(Me,Me)pro]-OH (20 mmole, 2 eq.) was coupled using HOBt (20 mmole, 2 eq) and DIC (20 mmole, 2 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF. Stage-21: Synthesis of Fmoc-Gly12-Lys(boc)13-Ser[Psi(Me,Me)pro]14-Ile15- Gln16-Asp(tbu)17-Leu18-Arg(pbf)19-Arg(pbf)20-Arg(pbf)21-Glu(tbu)22-Leu23- Leu24-Glu(tbu)25-Lys(boc)26-Leu27-Leu28-Aib29-Lys(boc)30-Leu31-His(Trt)32- Thr(tbu)33-Ala34–Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Gly-OH (25 mmole, 2.5 eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF. Stage-22: Synthesis of Fmoc-Lys(boc)11-Gly12-Lys(boc)13- Ser[Psi(Me,Me)pro]14-Ile15-Gln16-Asp(tbu)17-Leu18-Arg(pbf)19-Arg(pbf)20- Arg(pbf)21-Glu(tbu)22-Leu23-Leu24-Glu(tbu)25-Lys(boc)26-Leu27-Leu28-Aib29- Lys(boc)30-Leu31-His(Trt)32-Thr(tbu)33-Ala34–Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Lys(boc)-OH (25 mmole, 2.5 eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF. Stage-23: Synthesis of Fmoc-Asp(tbu)10-Lys(boc)11-Gly12-Lys(boc)13- Ser[Psi(Me,Me) pro]14-Ile15-Gln16-Asp(tbu)17-Leu18-Arg(pbf)19-Arg(pbf)20- Arg(pbf)21-Glu(tbu)22-Leu23-Leu24-Glu(tbu)25-Lys(boc)26-Leu27-Leu28-Aib29- Lys(boc)30-Leu31-His(Trt)32-Thr(tbu)33-Ala34–Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Asp(tbu)-OH (25 mmole, 2.5 eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF. Stage-24: Synthesis of Fmoc-His(Trt)9-Asp(tbu)10-Lys(boc)11-Gly12- Lys(boc)13-Ser[Psi(Me,Me)pro]14-Ile15-Gln16-Asp(tbu)17-Leu18-Arg(pbf)19- Arg(pbf)20-Arg(pbf)21-Glu(tbu)22-Leu23-Leu24-Glu(tbu)25-Lys(boc)26-Leu27- Leu28-Aib29-Lys(boc)30-Leu31-His(Trt)32-Thr(tbu)33-Ala34–Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-His(Trt)-OH (25 mmole, 2.5 eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF. Stage-25: Synthesis of Fmoc-Leu8-His(Trt)9-Asp(tbu)10-Lys(boc)11-Gly12- Lys(boc)13-Ser[Psi(Me,Me)pro]14-Ile15-Gln16-Asp(tbu)17-Leu18-Arg(pbf)19- Arg(pbf)20-Arg(pbf)21-Glu(tbu)22-Leu23-Leu24-Glu(tbu)25-Lys(boc)26-Leu27- Leu28-Aib29-Lys(boc)30-Leu31-His(Trt)32-Thr(tbu)33-Ala34–Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Leu-OH (25 mmole, 2.5 eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF. Stage-26: Synthesis of Fmoc-Leu7-Leu8-His(Trt)9-Asp(tbu)10-Lys(boc)11- Gly12-Lys(boc)13-Ser[Psi(Me,Me)pro]14-Ile15-Gln16-Asp(tbu)17-Leu18- Arg(pbf)19-Arg(pbf)20-Arg(pbf)21-Glu(tbu)22-Leu23-Leu24-Glu(tbu)25- Lys(boc)26-Leu27-Leu28-Aib29-Lys(boc)30-Leu31-His(Trt)32-Thr(tbu)33-Ala34– Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Leu- OH (25 mmole, 2.5 eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF. Stage-27: Synthesis of Fmoc- Gln6-Leu7-Leu8-His(Trt)9-Asp(tbu)10- Lys(boc)11-Gly12-Lys(boc)13-Ser[Psi(Me,Me)pro]14-Ile15-Gln16-Asp(tbu)17- Leu18-Arg(pbf)19-Arg(pbf)20-Arg(pbf)21-Glu(tbu)22-Leu23-Leu24-Glu(tbu)25- Lys(boc)26-Leu27-Leu28-Aib29-Lys(boc)30-Leu31-His(Trt)32-Thr(tbu)33-Ala34– Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Gln- OH (25 mmole, 2.5 eq.) was coupled using HOBt (50 mmole, 5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF. Stage-28: Synthesis of Fmoc-His(Trt)5-Gln6-Leu7-Leu8-His(Trt)9-Asp(tbu)10- Lys(boc)11-Gly12-Lys(boc)13-Ser[Psi(Me,Me)pro]14-Ile15-Gln16-Asp(tbu)17- Leu18-Arg(pbf)19-Arg(pbf)20-Arg(pbf)21-Glu(tbu)22-Leu23-Leu24-Glu(tbu)25- Lys(boc)26-Leu27-Leu28-Aib29-Lys(boc)30-Leu31-His(Trt)32-Thr(tbu)33-Ala34– Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc- His(Trt)-OH (25 mmole, 2.5 eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF. Stage-29: Synthesis of Fmoc-Glu(tbu)4-His(Trt)5-Gln6-Leu7-Leu8-His(Trt)9- Asp(tbu)10-Lys(boc)11-Gly12-Lys(boc)13-Ser[Psi(Me,Me)pro]14-Ile15-Gln16- Asp(tbu)17-Leu18-Arg(pbf)19-Arg(pbf)20-Arg(pbf)21-Glu(tbu)22-Leu23-Leu24- Glu(tbu)25-Lys(boc)26-Leu27-Leu28-Aib29-Lys(boc)30-Leu31-His(Trt)32- Thr(tbu)33-Ala34–Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Glu(tbu)-OH (25 mmole, 2.5 eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF. Stage-30: Synthesis of Fmoc-Ser(tbu)3-Glu(tbu)4-His(Trt)5-Gln6-Leu7-Leu8- His(Trt)9-Asp(tbu)10-Lys(boc)11-Gly12-Lys(boc)13-Ser[Psi(Me,Me)pro]14-Ile15- Gln16-Asp(tbu)17-Leu18-Arg(pbf)19-Arg(pbf)20-Arg(pbf)21-Glu(tbu)22-Leu23- Leu24-Glu(tbu)25-Lys(boc)26-Leu27-Leu28-Aib29-Lys(boc)30-Leu31-His(Trt)32- Thr(tbu)33-Ala34–Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Ser(tbu)-OH (25 mmole, 2.5 eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF. Stage-31: Synthesis of Fmoc-Val2-Ser(tbu)3-Glu(tbu)4-His(Trt)5-Gln6-Leu7- Leu8-His(Trt)9-Asp(tbu)10-Lys(boc)11-Gly12-Lys(boc)13-Ser[Psi(Me,Me)pro]14- Ile15-Gln16-Asp(tbu)17-Leu18-Arg(pbf)19-Arg(pbf)20-Arg(pbf)21-Glu(tbu)22- Leu23-Leu24-Glu(tbu)25-Lys(boc)26-Leu27-Leu28-Aib29-Lys(boc)30-Leu31- His(Trt)32-Thr(tbu)33-Ala34–Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Val-OH (25 mmole, 2.5 eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF. Stage-32: Synthesis of Fmoc-Ala1-Val2-Ser(tbu)3-Glu(tbu)4-His(Trt)5-Gln6- Leu7-Leu8-His(Trt)9-Asp(tbu)10-Lys(boc)11-Gly12-Lys(boc)13- Ser[Psi(Me,Me)pro]14-Ile15-Gln16-Asp(tbu)17-Leu18-Arg(pbf)19-Arg(pbf)20- Arg(pbf)21-Glu(tbu)22-Leu23-Leu24-Glu(tbu)25-Lys(boc)26-Leu27-Leu28-Aib29- Lys(boc)30-Leu31-His(Trt)32-Thr(tbu)33-Ala34–Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Ala-OH (25 mmole, 2.5 eq.) was coupled using HOBt (25 mmole, 2.5 eq) and DIC (25 mmole, 2.5 eq) in DMF solvent. The mixture was stirred in the presence of Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF. Stage-33: Synthesis of H-Ala1-Val2-Ser(tbu)3-Glu(tbu)4-His(Trt)5-Gln6-Leu7- Leu8-His(Trt)9-Asp(tbu)10-Lys(boc)11-Gly12-Lys(boc)13-Ser[Psi(Me,Me)pro]14- Ile15-Gln16-Asp(tbu)17-Leu18-Arg(pbf)19-Arg(pbf)20-Arg(pbf)21-Glu(tbu)22- Leu23-Leu24-Glu(tbu)25-Lys(boc)26-Leu27-Leu28-Aib29-Lys(boc)30-Leu31- His(Trt)32-Thr(tbu)33-Ala34–Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. Post completion of the synthesis, the resin was thoroughly washed with DMF, DCM and Et2O and drying of resin in desiccator.
Weight of the peptidyl resin: 80 gm Stage-34: Preparation of Crude Abaloparatide: H-Ala1-Val2-Ser3-Glu4-His5- Gln6-Leu7-Leu8-His9-Asp10-Lys11-Gly12-Lys13-Ser14-Ile15-Gln16-Asp17-Leu18- Arg19-Arg20-Arg21-Glu22-Leu23-Leu24-Glu25-Lys26-Leu27-Leu28-Aib29-Lys30- Leu31-His32-Thr33-Ala34–NH2:Simultaneous deprotection of all the protecting groups was carried out by the treatment of either of the cocktail cleavage mixtures stated below: TFA:EDT:Phenol:Thioanisole:water (82.5:2.5:5:5:5 v/v) or TFA:TIS:DTT:Water (85:5:5:5 v:w/v) preferably with TFA:EDT: Phenol:Thioanisole:water (82.5:2.5:5:5:5 v/v) cocktail mixture i.e K type reagent. The cleavage was carried out at <10°C for initial 15 minutes followed by the stirring of the peptidyl resin for 3 hours at ambient temperature. The crude cleavage mixture was then filtered, the resin washed thoroughly with TFA. The filtrate was dropped on to 12 mL of cold dry Diethyl ether per mL of cocktail and further 6 additional washing with 0.5 Liter of diethyl ether were done to the product. Product was dried under vacuum for 16 hrs.
The isolated yield of the crude peptide: 40 gm (100% process yield)
The Purity of the crude peptide: 83%
HPLC chromatogram of Crude Abaloparatide acetate is depicted in FIG 2. Example 3: Preparation method of Abaloparatide linear peptide resin on rink amide resin of functionality 0.52 mmol/g.
The process is same as described in the Example 1, and only the change is in functionality of Initial rink MBHA amide resin i.e. rink amide resin of functionality 0.52 mmol/gm (standard substitution value of 0.50-0.60 mmole/g) employed for the synthesis. (Process yield 100 %, purity 45 %).
HPLC chromatogram of Crude Abaloparatide acetate is depicted in FIG 3. Example 4: Purification of the Crude Abaloparatide
Crude Abaloparatide was purified by reverse phase C-8 HPLC using TEAP buffer (as buffer A) and Acetonitrile (as buffer B). Post purification, the fractions were analysed for the purity. The fraction containing >97% were pooled and were further taken up for salt exchange (desalting). The salt exchange was performed on RP-HPLC using ammonium acetate. The fractions containing >99% were pooled and were lyophilized to isolate pure Abaloparatide which is further stored at -20°C.
The overall isolated purification yield was 24%.
HPLC chromatogram of purified Abaloparatide acetate is depicted in FIG.4 and mass is depicted in FIG.5. Example 5: Preparation method of Abaloparatide linear peptide resin on rink amide resin of functionality 0.33 mmol/g (without using pseudoproline Fmoc-Lys(Boc) Ser[Psi(Me,Me)Pro]-OH unit): The process is same as described in the Example 1, and the only change is that “No pseudoproline dipeptide unit is employed in the synthesis”. (Process yield 80 %, purity 37 %).HPLC chromatogram of Crude Abaloparatide acetate is depicted in FIG 6. Advantages of the present invention:
1. Reduced aggregation of peptides in the peptide chain is advocated to the use of Fmoc-Lys(boc)-Ser[Psi(Me,Me)Pro]-OH.
2. Use of Fmoc-Leu-Aib-OH reduces possible deletion impurities which can be formed due to incomplete coupling of Fmoc-Leu28-OH after Fmoc-Aib29- OH Coupling to the peptide chain bound to the resin in SPPS.
3. The use of rink amide resin of low substitution value of 0.1 to 0.4 mmol/g in the present invention affords the crude Abaloparatide having purity in a range of 80% to 85% compared to synthesis of Abaloparatide when performed on rink amide resin of standard substitution value which results in crude Abaloparatide of very low quality of approximately 45% purity.
4. An increased purity of the crude Abaloparatide renders the further downstream processing to be convenient, less strenuous and reduces the cost involved.
Figure imgf000029_0001
SEQUENCE LISTING <110> Hemmo Pharmaceuticals Pvt. Ltd.
<120> A process for preparing Abaloparatide <140>
<141>
<150> Indian Patent Application No.2019210___ <151> 10/04/2019 <160> 1
<210> 1
<211> 34
<212> Protein
<213> Artificial sequence
<220>
<223> Amino acid sequence <400> 1
Ala Val Ser Glu His Gln Leu Leu His Asp
Lys Gly Lys Ser Ile Gln Asp Leu Arg Arg
Arg Glu Leu Leu Glu Lys Leu Leu Aib Lys
Leu His Thr Ala

Claims

We claim, 1. A process for the synthesis of Abaloparatide represented by Seq Id No.1 comprising;
(i) elongation of a peptide with sequential addition of protected amino acid(s) and dipeptide units by solid phase synthesis to a solid support to obtain a protected peptide bound to a support,
(ii) cleaving and de-protecting the resin simultaneously to obtain crude Abaloparatide followed by purification to obtain purified Abaloparatide; and
(iii) isolation of Abaloparatide.
wherein the a-amino group of the amino acid/dipeptide is protected by Fmoc,
wherein a peptide bond is formed between a carboxyl group of the Fmoc protected amino acid/dipeptide unit and the amino group lined to the support.
2. The process as claimed in claim 1, wherein the Fmoc protected dipeptide is Fmoc-Lys(boc)-Ser[Psi(Me,Me)Pro]-OH and Fmoc-Leu-Aib-OH.
3. The process as claimed in claim 1, wherein the solid support is selected from Rink amide resin, Rink amide AM resin, Rink amide BHA resin or Sieber resin and Rink amide MBHA resin.
4. The process as claimed in claim 3, wherein the said Rink amide resin is having substitution value of 0.1 mmole/gm to 0.4 mmole/gm.
5. The process as claimed in claim 1, wherein elongation of peptide is carried out in the presence of a coupling agent (s).
6. The process as claimed in claim 1, wherein the coupling agent (s) is selected from the group comprising hydroxybenzotriazole (HOBt), N, N'- diisopropylcarbodiimide (DIC), O-(Benzotriazol-1-yl)-N,N,N',N'- tetramethyluronium tetrafluoroborate (TBTU), N,N,N¢,N¢-Tetramethyl-O- (1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU), 1,3- dicyclohexylcarbodlimide (DCC), 1-(dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (EDC HCl), benzotriazol-1-yl-oxy- tris(dimethyl-amino)-phosphonium hexafluorophosphate (BOP), N,N-bis- (2-oxo-3-oxazolidinyl)phosphonic dichloride (BOP-C1), benzotriazol-1- yloxytri(pyrrolidino)phosphonium hexafluorophosphate (PyBOP), bromotri(pyrrolidino)phosphonium hexafluorophosphate (PyBrOP), chlorotri(pynolidino)phosphonium hexafluorophosphate (PyClOP), ethyl- 2-cyano-2-(hydroxyimino) acetate (Oxyma Pure), O-(6-Chloro-1- hydrocibenzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TCTU), 245-norbornen-2,3-dicarboximido)-1,1,3,3-tetramethyluronium tetrafluoroborate (TNTU), 2-succinimido-1,1,3,3-tetramethyluronium tetrafluoro borate (TSTU), 1-Cyano-2-ethoxy-2- oxoethylidenaminooxy)dimethylamino-morpholino-carbenium
hexafluorophosphate (COMU), 3-(diethoxyphosphoryloxy)-1,2,3- benzotriazin-4(3H)-one (DEPBT) or mixtures thereof.
7. The process as claimed in claim 1, wherein the coupling takes place in presence of a solvent selected from the group comprising of DMF, DCM, THF, NMP, DMAC methanol, ethanol, isopropanol, dichloroethane, 1, 4- dioxane, ethyl acetate, acetonitrile, acetone, and the like or a mixtures thereof.
8. The process as claimed in claim 1, wherein Fmoc is removed from the peptide in the presence of an organic base prepared in an organic solvent.
9. The process as claimed in claim 1, wherein the organic base is piperidine, piperazine, N-methyl morpholine, diethyl amine, triethyl amine, 1,8- Diazabicyclo [5.4.0]undec-7-ene (DBU) and the like or a mixtures thereof.
10. The process as claimed in claim 1, wherein the organic solvent is dimethyl formamide (DMF), N-Methyl-2-Pyrrolidone (NMP), dichloromethane (DCM), tetrahydrofuran (THF), N,N-dimethylacetamide (DMAC) and the like or a mixtures thereof.
11. The process as claimed in claim 1, wherein the deprotection of the peptide from the resin is carried out in the presence of combination of Trifluoroacetic acid (TFA) and radical scavenger(s).
12. The process as claimed in claim 10, wherein the radical scavenger(s) is selected from the group comprising of triisopropylsilane (TIS), dithiothreitol (DTT), 1,2-ethanedithiol (EDT), Phenol, cresol, thioanisole, ammonium iodide, DMS and water.
PCT/IN2020/050338 2019-04-11 2020-04-09 A process for preparing abaloparatide WO2020208650A1 (en)

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CN111944040A (en) * 2020-08-11 2020-11-17 海南中和药业股份有限公司 Method for solid-phase synthesis of abamectin
US11806387B1 (en) 2022-05-20 2023-11-07 Radius Health, Inc. Process of making abaloparatide

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CN106146648A (en) * 2015-03-26 2016-11-23 深圳翰宇药业股份有限公司 A kind of synthetic method of parathyroid hormone analogs
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Publication number Priority date Publication date Assignee Title
CN111944040A (en) * 2020-08-11 2020-11-17 海南中和药业股份有限公司 Method for solid-phase synthesis of abamectin
CN111944040B (en) * 2020-08-11 2023-07-18 海南中和药业股份有限公司 Solid phase synthesis method of abamectin
US11806387B1 (en) 2022-05-20 2023-11-07 Radius Health, Inc. Process of making abaloparatide

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