DESC:FIELD OF THE INVENTION

The present invention relates to a process for the preparation of human parathyroid hormone related peptide analog abaloparatide. More particularly the present invention relates to the Solid Phase Process Synthesis (SPPS) comprising elongation of peptide followed by sequential coupling of protected amino acids and purification of the obtained abaloparatide.

BACKGROUND OF THE INVENTION

Abaloparatide is a human parathyroid hormone related peptide [PTHrP(1-34)] analog represented by formula: 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-NH2. Abaloparatide is 34 amino acid synthetic analog of PTHrP. It has 41% homology to parathyroid hormone (PTH) (1-34) and 76% homology to parathyroid hormone-related protein (PTHrP) (1-34).

It works as an anabolic agent for the bone, through selective activation of the parathyroid hormone 1 receptor (PTH1R), a G protein-coupled receptor (GPCR) expressed in the osteoblasts and osteocytes. Abaloparatide preferentially binds the RG conformational state of the PTH1R, which in turn elicits a transient downstream cyclic AMP signaling response towards to a more anabolic signaling pathway.

WO97/02834 describes the synthesis of human PTH (1-34) and PTHrP analogues using Solid Phase Peptide Synthesis (SPPS), which involves step-by-step addition of amino acids which are Boc-protected at the amino terminus. This method of peptide synthesis requires repeated deprotection cycles with strong acids such as trifluoroacetic acid (TFA), for the removal of the Boc-protecting group at each addition step. In addition to this, the process requires the use of Hydrogen Fluoride (HF) for final cleavage of the peptide from the resin. Hydrogen fluoride is a highly toxic and corrosive compound and its use is not recommended in large scale production of pharmaceutical compounds. As an overall consequence, these drawbacks result in an extremely difficult and environment unfriendly process.

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.

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.

In view of the above, the present invention aims to provide novel method for synthesizing Abaloparatide such that, the method of the present invention significantly reduces the maximum single impurity and improves the purity of Abaloparatide on the premise of ensuring the total yield of the Abaloparatide.

SUMMARY OF THE INVENTION

The primary objective of the present invention is to provide solid phase peptide synthesis process for the preparation of Abaloparatide. More particularly, the process for the preparation of abaloparatide which is an analog of parathyroid hormone related peptide. The process of the present invention results in a product with high yield and high purity. The process comprising steps:
i elongation of a peptide with sequential addition of protected amino acids to a solid support;
ii cleaving and de-protecting the resin at the same time to obtain crude abaloparatide; and
iii purification to obtain purified abaloparatide.

In one aspect, the present invention relates to the process for the preparation of abaloparatide through sequentially coupling protected amino acids.

In another aspect, the protected amino acids are coupled using HOBt.H2O/Oxyma and DIPC in DMF.

In yet another aspect, there is provided an improved process for preparing abaloparatide having purity >99% after purification with any individual impurity <0.5%.

Yet another objective of the present invention is to reduce the maximum single impurity and improve the purity of abaloparatide on the premise of ensuring the total yield of the abaloparatide.

BRIEF DESCRIPTION OF DRAWINGS
Figure 1: Depicts mass chromatogram of abaloparatide.
Figure 2: Depicts HPLC Chromatogram of Abaloparatide.

DETAILED DESCRIPTION OF THE INVENTION
Abbreviations:
Fmoc: fluorenylmethyloxycarbonyl or 9-fluorenylmethoxycarbonyl
Fmoc: AA: the amino acid of fluorenylmethyloxycarbonyl protection
BOC: tert-butoxycarbonyl protecting group
DMF: N, N-Dimethylformamide
Cbz: Benzyl chloroformate
Bpoc: 2-(p-biphenylyl)-2-propyloxycarbonyl
HOBt: N-hydroxybenzotriazole
DIPC: N, N'-Diisopropylcarbodiimide
TFA: trifluoroacetic acid
EDT: Ethanedithiol
His: histidine
HPLC: high performance liquid chromatography
Ala: alanine
Ile: isoleucine
Gln: glutamine
Glu: glutamic acid
Asn: asparagine
Cys: halfcystine
Leu: leucine
Lys: lysine
Phe: phenylalanine
Ser: serine
Trp: tryptophan
Tyr: tyrosine
Val: valine
MBHA resin: Methylbenzylhydrylamine resin
Gly: glycine
Trt: trityl
Me: methyl
Piperidine: hexahydropyridine

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 a peptide with sequential addition of protected amino acids to a solid support.

In a preferred embodiment, the present invention provides a process for the synthesis of abaloparatide comprising;
i elongation of a peptide with sequential addition of protected amino acids to a solid support;
ii cleaving and de-protecting the resin at the same time to obtain crude abaloparatide; and
iii purification to obtain purified abaloparatide.

The inter- or intra-molecular ß-sheet interactions occur during peptide synthesis using both solid-phase and solution-phase methodologies and they are stabilized and mediated by non-covalent hydrogen bonds, which—depending on the sequence—are favoured. These interactions arise on the backbone of the peptide, between the hydrogen amides and the carbonyls. The tendency of peptide chains to aggregate is translated into a list of common behavioural features attributed to "difficult sequences". The main relevant synthetic difficulties are the following: repetitive incomplete amino acylations despite re-couplings; accentuated difficulties when resin loading is high or when sterically hindered amino acids (AAs) are present in the sequence; and more importantly, slow, or incomplete 9-fluorenylmethoxycarbonyl removal.

Majorly in case of abaloparatide, during long peptide synthesis, aggregation occurs more commonly due to static charge present in the electronegative parts. The aggregation is manifested not only during the synthesis of peptides on solid phase, but also when the sequence is unprotected in solution. The presence of alanine which show a notable capacity to assemble. The chain elongation of the long-chain peptide like abaloparatide get hindered and resulted into the incomplete synthesis. To avoid such difficulties, selection of resin in case of abaloparatide is most important. The resin with loading capacity 0.3 to 0.4 mmol is most suitable for the complete synthesis. There is an contraction of peptide bed height after 19th amino acid sequence (Fmoc-Arg (Pbf)-OH;19AA). Further the elongation of peptide chain hindered due to consecutive addition of three Fmoc-Arg (Pbf)-OH amino acid. Finally, the progress of peptide chain gets almost terminated. After modification of several factors like higher concentration of reagents, changing the solvent or moderation of reaction conditions, it was found that wash with 1% 1-hydroxybenzotriazole monohydrate (HOBT.H2O) in DMF eliminates the effect of resin aggregation. The elimination of aggregation of resin lead to moderate the coupling of long chain peptide in case of abaloparatide. This is again ascertained by the physical observation of heights of the resin beds in peptide vessel. The washing with 1-hydroxybenzotriazole monohydrate (HOBT.H2O) in DMF significantly increase the height of the deprotected peptide resin bed. Increase the bed height further improves the coupling of the progressive amino acid sequence.

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 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 Fmoc protection or Boc 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 unit is Fmoc.

In a preferred embodiment, the solid support resin is selected from Rink amide AM resin having loading capacity 0.4-0.6 mmole/g or Rink amide MBHA resin having loading capacity 0.3-0.5 mmole/g.

The resin is deprotected with 20% piperidine in DMF. Next, the mixture of Fmoc-Ala-OH, HOBt.H2O and DIPC in DMF is put into the vessel for first amino acid coupling. Resin is then stirred for around 2 hours till Kaiser Test became negative to determine the completeness of the coupling reaction. Then after, resin is washed and de-protected from Fmoc for the sequential couplings.

Subsequently, Fmoc-Thr(tBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Aib-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Glu(OtBu), Fmoc-Ser(tBu)-OH, Fmoc-Val-OH and Fmoc-Ala-OH are coupled sequentially using HOBt.H2O/Oxyma and DIPC in DMF. The peptide resin is washed and dried after de-protection.

Further, in a separate clean and dry RBF, charge mixture of Trifluoroacetic acid (TFA), Ethane dithiol (EDT), phenol and water and then it is cooled to 0-5oC. The reaction mass is kept stirring till cleavage and total de-protection of the peptide gets completed. The reaction mass is then filtered under vacuum and the filtrate is distilled. Di-isopropyl ether is added into the RBF to get solid mass which is filtered under vacuum. The solid mass is dried to get Abaloparatide crude having 60-70% purity.

In a preferred embodiment, crude abaloparatide is further purified by preparative HPLC. In the first step, preparative column is packed with 5µm C18 and is saturated with buffer. Abaloparatide is purified by preparative HPLC using a combination of a buffer and an organic solvent. More preferably, the said combination comprises ammonium acetate buffer and Acetonitrile. Next, abaloparatide crude is dissolved in water and injected. A gradient of ammonium acetate buffer and acetonitrile are used as mobile phase. Finally, abaloparatide is eluted in fractions. Post purification, the fractions are analyzed for their purity. The obtained abaloparatide is >99% pure with any individual impurity <0.5%. The salt exchange (desalting) is performed on HPLC itself using ammonium acetate and then lastly abaloparatide is lyophilized and stored.
In a preferred embodiment, the present is described schematically in the following manner:
Stage-I: Synthesis of abaloparatide crude

Stage-II: Purification of crude abaloparatide

Stage-III: Desalting of pure abaloparatide


EXAMPLE: 1: Spectrophotometric analysis of Abaloparatide.

Analysis molecular mass of abaloparatide (M) is 3959.64. The mass chromatogram of the compound is depicted in Figure 1. From mass chromatogram, the observed fragments were 1980.5 (M-1)/2, 1320.1(M-1)/3 in negative mode and 1982.2 (M+2)/2, 1322.3 (M+2)/3, 991.6 (M+2)/4 in positive mode where M is the molecular weight of the abaloparatide. In analysis through High Resolution Mass spectroscopy of Abaloparatide performed using a 10-minute reverse phase separation coupled to HRMS-QTOF LC-MS/MS. Intact mass of abaloparatide is observed to be 3958.27 Da.

EXAMPLE: 2: Chromatographic analysis of Abaloparatide.

Analysis molecular mass of abaloparatide performed in HPLC. The analysis is based on sample concentration of 1000ppm with water as a diluent. The chromatogram obtained is depicted in Figure 2.
,CLAIMS:CLAIMS
We claim;
[CLAIM 1]. A process for synthesis of abaloparatide an parathyroid hormone analog comprises:
i elongation of a peptide with sequential addition of protected amino acids to a solid support comprises steps of:
a. treating solid support resin with 20% piperidine in N and N-Dimethylformamide [DMF] in peptide vessel;
b. addition of 9-fluorenylmethoxycarbonyl protected alanine [Fmoc-Ala-OH], N-hydroxybenzotriazole [HOBt.H2O] and N, N'-Diisopropylcarbodiimide [DIPC] in DMF into the peptide vessel for amino acid alanine coupling;
c. stirring resin for at least 2 hours till Kaiser Test becomes negative;
d. washing and deprotecting resin from Fmoc for successive couplings;
e. coupling sequentially 9-fluorenylmethoxycarbonyl protected amino acids Fmoc-Thr(tBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Aib-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Ser (tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Glu(OtBu), Fmoc-Ser(tBu)-OH, Fmoc-Val-OH and Fmoc-Ala-OH using HOBt.H2O/Oxyma and DIPC in DMF;

ii cleaving and de-protecting the resin to obtain crude abaloparatide comprises steps of:
a. treating charge mixture of Trifluoroacetic acid (TFA), Ethanedithiol (EDT), Phenol and water in a separate clean and dry (Round Bottom Flask) RBF;
b. cooling the treated mixture at 0-5oC;
c. stirring till cleavage and total deprotection of the peptide gets completed;
d. filtering under vacuum and the distillation of the filtrate;
e. addition of diisopropyl ether into the RBF to get solid mass filtered under vacuum;
f. drying the solid mass to get Abaloparatide crude having 60-70% purity; And

iii Purification through preparative HPLC using mobile phase to obtain purified abaloparatide followed by spectrophotometric analysis of the same.

[CLAIM 2]. The process as claimed in claim 1, wherein solid support resin is selected from Rink amide AM resin having loading capacity 0.4-0.6 mmole/g, Rink amide Methylbenzylhydrylamine [MBHA] resin having loading capacity 0.3-0.5 mmole/g.

[CLAIM 3]. The process as claimed in claim 1, wherein amino acid protecting group is selected from the group consisting of fluorenylmethyloxycarbonyl, [Fmoc], tert-butoxycarbonyl [BoC] Benzyl chloroformate [Cbz], 2-(p-biphenylyl)-2-propyloxycarbonyl [Bpoc].

[CLAIM 4]. The process as claimed in claim 1, wherein purification through preparative HPLC column is packed with 5µm C18 and is saturated with ammonium acetate buffer.

[CLAIM 5]. The process as claimed in claim 1, wherein mobile phase is gradient of ammonium acetate buffer and acetonitrile.

[CLAIM 6]. The process as claimed in claim 1, wherein analysis mass chromatogram of purified abaloparatide obtained from the process, the observes fragments at 1980.5 (M-1)/2, 1320.1(M-1)/3 in negative mode and 1982.2 (M+2)/2, 1322.3 (M+2)/3, 991.6 (M+2)/4 in positive mode.

Dated this 26th day of July, 2022